Humanized antibodies against CD269 (BCMA)

ABSTRACT

A nucleic acid molecule encoding an antibody or antibody fragment, wherein the antibody or antibody fragment binds an epitope of the extracellular domain of CD269 (BCMA), a host cell comprising the nucleic acid molecule and a composition comprising the host cell.

The invention relates to humanized antibodies or antibody fragments thatbind CD269 (BCMA), thereby disrupting the interaction between CD269 andits native ligands (BAFF and APRIL), and their use in the treatment ofplasma cell-mediated diseases such as multiple myeloma and autoimmunediseases.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing is 33754389_1. TXT,the date of creation of the ASCII text file is Oct. 23, 2020, and thesize of the ASCII text file is 53.8 KB.

BACKGROUND OF THE INVENTION

The B cell maturation antigen (BCMA) is member 17 of the tumor necrosisfactor receptor superfamily (TNFRSF). Its native ligands are the B cellactivating factor (BAFF; also called BLyS or TALL-1, TNFSF13B) and aproliferation-inducing ligand (APRIL, TNFSF13, CD256) which are involved(through interaction with further ligands) in regulating various aspectsof humoral immunity, B cell development, and homeostasis.

BCMA is highly expressed on malignant plasma cells, for example inmultiple myeloma, (MM), which is a B cell non-Hodgkin lymphoma of thebone marrow, and plasma cell leukemia (PCL), which is more aggressivethan MM and constitutes around 4% of all cases of plasma cell disorders.In addition to MM and PCL, BOMA has also been detected on Hodgkin andReed-Sternberg cells in patients suffering from Hodgkin's lymphoma (Chiuet al. (2007) Blood 109:729-739). Similar to its function on plasmacells, ligand binding to BCMA has been shown to modulate the growth andsurvival of multiple myeloma cells expressing BCMA (Novak et al. (2004)Blood 103:689-694). Signalling of BAFF and APRIL via BCMA are consideredas pro-survival factors for malignant plasma cells; hence, the depletionof BCMA-positive tumour cells and/or the disruption of ligand-receptorinteraction should improve the therapeutic outcome for multiple myelomaand autoantibody-dependent autoimmune diseases.

There are presently various approaches available for the treatment ofmultiple myeloma (Raab et al. (2009) Lancet 374:324-339). Chemotherapyleads in most subjects only to partial control of multiple myeloma; onlyrarely does chemotherapy lead to complete remission. Combinationapproaches are therefore often applied, commonly involving an additionaladministration of corticosteroids, such as dexamethasone or prednisone.Corticosteroids are however plagued by side effects, such as reducedbone density. Stem cell transplantation has also been proposed, usingone's own stem cells (autologous) or using cells from a close relativeor matched unrelated donor (allogeneic). In multiple myeloma, mosttransplants performed are of the autologous kind. Such transplants,although not curative, have been shown to prolong life in selectedpatients (Suzuki (2013) Jpn J Clin Oncol 43:116-124). Alternativelythalidomide and derivatives thereof have recently been applied intreatment but are also associated with sub-optimal success rates andhigh costs. More recently, the proteasome inhibitor bortezomib (PS-341)has been approved for the treatment of relapsed and refractory MM andwas used in numerous clinical trials alone or in combination withestablished drugs resulting in an encouraging clinical outcome(Richardson et al. (2003) New Engl J Med 348:2609-2617; Kapoor et al.(2012) Semin Hematol 49:228-242). The costs for combined treatments arecorrespondingly high and success rates still leave significant room forimprovement. The combination of treatment options is also not ideal dueto an accumulation of side effects if multiple medicaments are usedsimultaneously. Novel approaches for the treatment of plasma celldiseases, in particular multiple myeloma, are required.

The ability to specifically target plasma cells is also of great benefitfor the treatment of autoimmune diseases. Mild forms of autoimmunedisease are usually initially treated with nonsteroidalanti-inflammatory drugs (NSAID) or disease-modifying anti-rheumaticdrugs (DMARD). More severe forms of Systemic Lupus Erythematosus (SLE),involving organ dysfunction due to active disease, usually are treatedwith steroids in conjunction with strong immunosuppressive agents suchas cyclophosphamide, a cytotoxic agent that targets cycling cells. Onlyrecently Belimumab, an antibody targeting the cytokine BAFF, which isfound at elevated levels in serum of patients with autoimmune diseases,received approval by the Food and Drug Administration (FDA) for its usein SLE. However, only newly formed B cells rely on BAFF for survival inhumans, whereas memory B cells and plasma cells are less susceptible toselective BAFF inhibition (Jacobi et al. (2010) Arthritis Rheum62:201-210). For rheumatoid arthritis (RA), TNF inhibitors were thefirst licensed biological agents, followed by abatacept, rituximab, andtocilizumab and others: they suppress key inflammatory pathways involvedin joint inflammation and destruction, which, however, comes at theprice of an elevated infection risk due to relative immunosuppression(Chan et al. (2010) Nat Rev Immunol 10:301-316, Keyser (2011) CurrRheumatol Rev 7:77-87). Despite the approval of these biologicals,patients suffering from RA and SLE often show a persistence ofautoimmune markers, which is most likely related to the presence oflong-lived, sessile plasma cells in bone marrow that resist e.g.CD20-mediated ablation by rituximab and high dosage glucocorticoid andcyclophosphamid therapy.

Antibodies which bind CD269 (BCMA) and their use in the treatment ofvarious B-cell related medical disorders have been described in the art.Ryan et al (Molecular Cancer Therapeutics, 2007 6(11), 3009) describe ananti-BCMA antibody obtained via vaccination in rats using a peptide ofamino acids 5 to 54 of the BCMA protein. WO 2012/163805 describes BCMAbinding proteins, such as chimeric and humanized antibodies, their useto block BAFF and/or APRIL interaction with BCMA and their potential usein treating plasma cell malignancies such as multiple myeloma. Theantibody disclosed therein was obtained via vaccination in mouse using arecombinant peptide of amino acids 4 to 53 of the BCMA protein. WO2010/104949 also discloses various antibodies that bind preferably theextracellular domain of BCMA and their use in treating B cell mediatedmedical conditions and disorders. WO 2002/066516 and WO 2012/066058disclose bivalent antibodies that bind both BCMA and additional targetsand their potential use in the treatment of B cell related medicaldisorders. Details regarding the binding properties and specificepitopes of the bivalent antibodies are not provided in eitherdisclosure.

SUMMARY OF THE INVENTION

In light of the prior art the technical problem underlying the inventionwas the provision of an agent suitable for treating human diseasesassociated with pathogenic plasma cells, such as multiple myeloma andautoimmune diseases. This problem is solved by the features of theindependent claims. Preferred embodiments of the present invention areprovided by the dependent claims.

Therefore, an object of the invention is to provide a humanized antibodyor antibody fragment that binds CD269 (BCMA), in particular an epitopeof the extracellular domain of CD269 (BCMA).

The antibodies disclosed herein comprise humanized sequences, especiallyof the preferred VL and VH binding regions, which maintain theappropriate ligand affinities as described with respect to the chimericantibody J22.9-xi.

In various embodiments of the invention the amino acid sequencemodification to obtain said humanized sequences may occur in either theCDR regions of the original chimeric antibody J22.9-xi or in theframework regions, wherein the framework region is to be understood as aregion in the variable domain of a protein which belongs to theimmunoglobulin superfamily, and which is less “variable” than the CDRs.

It was entirely surprising that the particular humanized sequencesprovided herein, preferably the CDR regions of the VL and VH regionsinvolved in binding, exhibit the specific and strong binding asdemonstrated in the experimental examples, and maintain the bindingcharacteristics of the original chimeric antibody J22.9-xi to such anextent as to maintain their desired therapeutic effect.

The invention therefore relates to an antibody or antibody fragment,comprising a VH domain that comprises CDR sequences of:

-   -   RYWX₁S (H-CDR1; SEQ ID NO. 15), wherein X₁: I, F, L, V, Y. C, G,        A, S, T);    -   EINPX₂X₃STINYAPSLKDK (H-CDR2; SEQ ID No. 16), wherein X₂X₃: SS,        NS, TS, GS, KS, RS, SD, SN, DE; and/or    -   SLYX₄DYGDAX₅DYW (H-CDR3; SEQ ID NO. 17), wherein X₄: Y, L, A, V,        F, I, W, and/or X₅: Y, L, F, I, V, A, C,        wherein said antibody or fragment thereof specifically binds an        epitope of the extracellular domain of CD269 (BCMA).

It was particularly surprising that the humanized antibodies describedherein, that exhibit sequence changes compared to the original chimericantibody, in particular sequence changes in the CDRs of said chimericantibody, maintain sufficient binding properties towards their targetfor therapeutic efficacy.

A skilled person would not have expected that the bindingcharacteristics of the humanized variants would be similar to theoriginal chimeric or mouse antibody. Considering the sequence changes inthe variable domains, in particular the CDRs, the beneficial bindingcharacteristics of the humanized sequences demonstrated herein areconsidered a surprising technical effect. The comparison in bindingcharacteristics between the partially and fully humanized antibodiesalso shows improvements in the fully humanized sequences. Thisrepresents an entirely unexpected result. The initial modifications tothe chimera (partially humanized) led to some loss in binding affinity.However, the introduction of further humanizations subsequently lead toenhanced binding, whereby the “fully humanized” sequences show similarbinding properties compared to the original chimera, thereby showing asurprising technical effect after having made such significant sequencemodifications without severe loss of binding affinity.

In a preferred embodiment the antibody or antibody fragment as describedherein is characterised in that the VH domain comprises the CDR sequenceRYWIS (SEQ ID NO. 18) or RYWFS (SEQ ID NO. 19).

In a preferred embodiment the antibody or antibody fragment as describedherein is characterised in that said VH domain comprises the CDRsequence EINPNSSTINYAPSLKDK (SEQ ID No. 20) or EINPSSSTINYAPSLKDK (SEQID No. 21).

In further embodiments of the invention amino acid 54 of the VH domainmay relate to any given amino acid or modified amino acid. As shown inthe examples below, potential glycosylation of the N amino acid at thisresidue does not significantly disrupt specific and strong binding ofthe antibody to the target epitope. In light of this information, theinvention relates to an antibody or antibody fragment comprising an CDR2sequence as described herein, wherein any given amino acid or modifiedamino acid may be present at amino acid 54 of the VH domain in the CDR2sequence.

In a preferred embodiment the antibody or antibody fragment as describedherein is characterised in that said VH domain comprises the CDRsequence SLYYDYGDAYDYW (SEQ ID NO. 22).

The invention further relates to an antibody or antibody fragment,comprising a VL domain that comprises CDR sequences of:

-   -   KASQSVX₁X₂NVA (L-CDR1; SEQ ID NO. 23), wherein X₁X₂: ES, SS, TS,        QS, HS, DH;    -   SASLRFS (L-CDR2; SEQ ID NO 24); and/or    -   QQYNNYPLTFG (L-CDR3; SEQ ID NO. 25),        wherein said antibody or fragment thereof specifically binds an        epitope of the extracellular domain of CD269 (BCMA).

Also with respect to the LC sequence, it was surprising that themodified sequence of the CDR3 sequence had no significant detrimentaleffect on binding to the BCMA target.

Furthermore, antibodies of the present invention also show unexpectedand beneficial stability characteristics when in solution, both whenisolated or purified, or in vitro, and in vivo, post administration,that would not have been expected from the sequence changes carried outto the original chimeric antibody.

In a preferred embodiment the antibody or antibody fragment as describedherein is characterised in that the VL domain comprises the CDR sequenceKASQSVDSNVA (SEQ ID NO. 26).

In a preferred embodiment the antibody or antibody fragment as describedherein comprises a VH domain that comprises the sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWX₁SWVRQAPGKGLVWVGEINPX₂X₃STINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYX₄DYGDAX₅DYWGQGTLVTVSS (SEQ IDNO. 4), wherein X₁: I, F, L, V, Y. C, G, A, S, T; X₂X₃: SS, NS, TS, GS,KS, RS, SD, SN, DE, preferably SS; X₄: Y, L, A, V, F, I, W; and X₅: Y,L, F, I, V, A, C.

In a preferred embodiment the antibody or antibody fragment as describedherein is characterised in that the antibody or fragment comprises a VHdomain that comprises the sequence according to SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO. 8 or SEQ ID NO. 9.

In a preferred embodiment the antibody or antibody fragment as describedherein comprises a VL domain that comprises the sequenceEIVMTQSPATLSVSPGERATLSCKASQSVX₁X₂NVAWYQQKPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELKR (SEQ ID NO. 12), whereinX₁X₂: ES, SS, TS, QS, HS, DH.

In a preferred embodiment the antibody or antibody fragment as describedherein comprises a VL domain that comprises the sequence according toSEQ ID NO. 14.

In a preferred embodiment the antibody or antibody fragment as describedherein comprises a VH domain that comprises the sequence according toSEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9 and a VL domainthat comprises the sequence according to SEQ ID NO. 14.

The invention further relates to an antibody or antibody fragment asdescribed herein comprising a VH domain, wherein said VH domaincomprises a sequence according toX₁VQLX₂X₃SGGGLVQPGGSLX₄LSCAASGX₅X₆FX₇X₈YWZ₁SWVRX₃APGKGLEWX₁₀GEINPZ₂SSTINYAPSLKX₁₁X₁₂FX₁₃ISRDNAKNTLYLQMX₁₄X₁₅X₁₆RX₁₇EDTAX₁₈YYCASLYYDYGDAZ₃DYWGQGTX₁₉VTVSS(SEQ ID No. 41), wherein X₁: Q, E; X₂: Q, V; X₃: Q, E; X₄: K, R; X₅: I,F; X₆: D, T; X₇: S, D; X₈: R, D; X₉: R, Q; X₁₀: I, V; X₁₁: D, G; X₁₂: K,R; X₁₃: I, T; X₁₄: S, N; X₁₅: K, S; X₁₆: V, L; X₁₇: S, A; X₁₈: L, V;X₁₉: S, L; and wherein at least one of Z₁: I, F, L, V, Y. C, G, A, S, T,preferably I or F; Z₂: S, N, T, G, K, R, D, preferably S and/or Z₃: Y,L, F, I, V, A, C, preferably Y; and wherein said antibody or fragmentthereof specifically binds an epitope of the extracellular domain ofCD269 (BCMA).

This embodiment encompasses various humanized antibodies, in particularthe VH sequences thereof, all variants defined by the advantageoushumanization carried out in the CDRs as described herein.

The invention further relates to an antibody or antibody fragment asdescribed herein comprising a VL domain, wherein said VL domaincomprises a sequence according toDIVMTQSX₁X₂X₃X₄X₅X₆SVGDX₇VX₈X₃TCKASQSVESNVAWYQQKPX₁₃QX₁₁PKX₁₂LIX₁₃SX₁₄X₁₅LRFSGVPARFX₁₆GSGSGTDFTLTISX₁₇LQSEDX₁₈AX₁₃YX₂₀CQQYNNYPLTFGAGTKLELKR (SEQ ID No. 42), wherein X₁: Q, P; X₂: R, A; X₃: F, T; X₄: M, L; X₅:T, S; X₆: T, V; X₇: R, E; X₈: S, T; X₉: V, L; X₁₀: R, G; X₁₁: S, A; X₁₂:A, L; X₁₃: F, Y; X₁₄: A, D; X₁₅: S, D; X₁₆: T, S; X₁₇: N, S; X₁₈: L, F;X₁₉: E, V; X₂₀: F, Y; and wherein said antibody or fragment thereofspecifically binds an epitope of the extracellular domain of CD269(BCMA). This embodiment encompasses various humanized antibodies, inparticular the VL sequences thereof, all variants defined by theadvantageous humanization carried out in the CDRs as described herein.

Preferred Embodiments Regarding Humanized Antibody Variants

As disclosed in detail herein, the sequence of the preferred embodimentsof the invention according to J22.9-xi was humanized in order to providea more compatible reagent for administration in human subjects. Varioushumanized sequence variants of J22.9-xi have been generated and testedfor their binding affinity and specificity to both human and cynomolgusCD269 (BCMA). The results from binding assays demonstrate that thehumanized sequences maintain the desired binding properties of thechimeric reagent J22.9-xi. In the below sequences the underlined regionsrepresent the CDRs or putative CDRs.

Preferred Embodiments Regarding Humanized VH Variants

Additional information is provided below on the humanized antibodysequence of the present invention.

Chimeric Sequence:

Hc mouse (SEQ ID No. 1):QVQLQQSGGGLVQPGGSLKLSCAASGIDFSRYWMSWVRRAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCASLY YDYGDAMDYWGQGTSVTVSS

The HC mouse sequence represents the variable region of the heavy chain(VH) originally developed for the chimeric antibody J22.9-xi, whichcomprises VL and VH domains obtained from a mouse antibody, capable ofbinding an epitope of the extracellular domain of CD269 (BCMA), and theVL and VH domains are fused to human CL and CH domains, respectively.

Partially Humanized Sequences:

HC partially humanized (SEQ ID No. 2):EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYWMSWVRQAPGKGLEWVGEINPDSSTINYAPSLKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLY YDYGDAMDYWGQGTLVTVSS

The HC partially humanized sequence represents a modified amino acidsequence (via amino acid substitutions) in comparison to the chimericantibody disclosed herein, whereby the VL and VH binding regions havebeen modified with respect to their sequence to make them more suitablefor administration in humans.

Humanized VH Sequence:

hHC01  (SEQ ID No. 3) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLVWVGEINPDSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLY YDYGDAMDYWGQGTLVTVSS

Humanized VH sequence with removal of post translational modificationmotifs:

hHC02  (SEQ ID No. 4) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWX₁SWVRQAPGKGLVWVG EINPX ₂ X ₃STINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYX ₄DYGDAX ₅DYWGQGTLVTVSS

Wherein:

X₁: I, F, L, V, Y. C, G, A, S, T, preferably I or F;

X₂X₃: SS, NS, TS, GS, KS, RS, SD, SN, DE, preferably SS;

X₄: Y, L, A, V, F, I, W, preferably Y; and/or

X₅: Y, L, F, I, V, A, C, preferably Y;

The “hHC01” and “hHC02” humanized sequences represent novel amino acidsequences that comprise sequence changes in comparison to both theoriginal chimeric sequence and the partially humanized sequencesdescribed herein.

The PTM mutations are intended to remove potentially detrimental posttranslational modification motifs from said proteins, whilst maintainingthe advantageous binding properties. The positions 1, 5, 6, 19, 27, 28,34, 39, 46, 48, 54, 69, 84, 85, 86, 88, 93, 107 and/or 115 of hHC01 andhHC02 are preferably mutated (substituted) in comparison to the originalchimeric sequence. The importance of the substitution relates primarilyto the resulting amino acid, not the originating amino acid. The changemay therefore also be carried out from the corresponding amino acid ofthe original chimeric amino acid or other variant, such as the partiallyhumanized sequences.

The following substitutions are novel in comparison to the chimeric (SEQID No 1) sequence:

-   -   the amino acid M34 of the HC (VH) sequence is substituted with        any amino acid, preferably I, L, F, V, Y. C, G, A, S, T;    -   the amino acid E46 of the HC (VH) sequence is substituted with        V;    -   the amino acids D54 and S55 of the HC (VH) sequence is        substituted with any amino acid combination, preferably SS, TS,        GS, KS, RS, SD, SN, DE;    -   the amino acid Y101 of the HC (VH) sequence is substituted with        any amino acid, preferably L, A, V, F, I, W; and/or    -   the amino acid M107 of the HC (VH) sequence is substituted with        any amino acid, preferably L, Y, F, I, V, A, C.

Sequences that May be Modified at Those Residues Required for DirectInteraction with BCMA:

hHC03 - modified amino acids involved ininteraction with BCMA (SEQ ID No 5): EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYX₁MX ₂ WVRQAPGKGLVX ₃V GX ₄INPDSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCA SX ₅ X ₆ X ₇DYGDX₈MDYWGQGTLVTVSS

Wherein Preferred Amino Acids are:

X₁: W, F, Y, preferred W;

X₂: S, T, N, Q, D, E, preferred S;

X₃: W, F, Y, preferred W;

X₄: E, Q, preferred E;

X₅: L, I, V, G, A, preferred L;

X₆: Y, X, preferred Y;

X₇: Y, F, L, I, V, M, preferred Y; and/or

X₈: A, G, V, preferred A.

The “hHC03” humanized sequence represents novel amino acid sequencesthat comprise amino acid sequence changes in comparison to both theoriginal chimeric sequence and the partially humanized sequence. Thesesequence changes are intended to reflect potential changes in the aminoacids that bind the BCMA target, which may be substituted, whilstmaintaining the advantageous binding properties. The importance of thesubstitution relates primarily to the resulting amino acid, not theoriginating amino acid. The change may therefore also be carried outfrom the corresponding amino acid of the original chimeric amino acid orother variant.

For example:

-   -   the amino acid W33 of the HC (VH) sequence is W, F, Y;    -   the amino acid S35 of the HC (VH) sequence is S, T, N, Q, D, E;    -   the amino acid W47 of the HC (VH) sequence is W, F, Y;    -   the amino acid E50 of the HC (VH) sequence is E, Q;    -   the amino acid L99 of the HC (VH) sequence is L, I, V, G, A;    -   the amino acid Y100 of the HC (VH) sequence is Y, X;    -   the amino acid Y101 of the HC (VH) sequence is Y, F, L, I, V, M;        and/or    -   the amino acid A106 of the HC (VH) sequence is A, G, V.

In general, any change to a CDR region made during humanization may alsobe considered as a feature of a CDR sequence when consideredindependently of the framework sequence as a whole. Such modified CDRsequences may be considered defining features of the present invention,either within or independent of their context in the entire frameworkregion described herein. For example, the CDR sequences identified byunderline in the hHC01 to hHC03 may be considered a defining feature ofthe invention independently of the surrounding framework sequence.

Specific Examples of Humanized HC (VH) Sequences:

hHC04 (SEQ ID NO 6): EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWISWVRQAPGKGLVWVGEINPNSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLY YDYGDAYDYWGQGTLVTVSShHC05 (SEQ ID NO 7): EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWFSWVRQAPGKGLVWVGEINPNSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLY YDYGDAYDYWGQGTLVTVSShHC06 (SEQ ID NO 8): EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWISWVRQAPGKGLVWVGEINPSSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLY YDYGDAYDYWGQGTLVTVSShHC07 (SEQ ID NO 9): EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWFSWVRQAPGKGLVWVGEINPSSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLY YDYGDAYDYWGQGTLVTVSS

Alignments:

A CLUSTAL W (1.83) multiple sequence alignment of the varioussubstituted positions within the HC sequence provides appropriatesequence comparisons in FIG. 8 . The “General sequence” represents an HCsequence, whereby each X represents a potential amino acid change to anygiven amino acid. Preferred amino acid substitutions are those describedabove for each of the potentially mutated positions.

Preferred Embodiments Regarding Humanized VL Variants

Chimeric Sequence:

LC mouse (SEQ ID No. 43):DIVMTQSQRFMTTSVGDRVSVTCKASQSVDSNVAWYQQKPRQSPKALIFSASLRFSGVPARFTGSGSGTDFTLTISNLQSEDLAEYFCQQYNNYPLTFGA GTKLELKR

The LC mouse sequence represents the variable region of the light chain(VL) originally developed for the chimeric antibody J22.9-xi, whichcomprises VL and VH domains obtained from a mouse antibody, capable ofbinding an epitope of the extracellular domain of CD269 (BCMA), and theVL and VH domains are fused to human CL and CH domains, respectively.

Partially Humanized Sequences:

LC partially humanized (SEQ ID NO 10):DIVMTQSPATLSVSVGDEVTLTCKASQSVDSNVAWYQQKPGQAPKLLIYSDDLRFSGVPARFSGSGSGTDFTLTISSLQSEDFAVYYCQQYNNYPLTFGA GTKLELKR

The LC partially humanized sequence represents a modified sequence (viaamino acid substitutions) in comparison to the chimeric antibodydisclosed in the examples of the present invention, whereby the VL andVH binding regions have been modified with respect to their sequence tomake them more suitable for administration in humans.

Humanized VL Sequence:

hLC01 (SEQ ID NO 11): EIVMTQSPATLSVSPGERATLSCKASQSVDSNVAWYQQKPGQAPRALIYS ASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGA GTKLELKR

Humanized VL Sequence with Removal of Post Translational ModificationMotifs:

hLC02 (SEQ ID NO 12): EIVMTQSPATLSVSPGERATLSCKASQSVX ₁ X₂NVAWYQQKPGQAPRALI YSASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELKR

Wherein:

X₁X₂: ES, SS, TS, QS, HS, DH, preferably ES.

The “hLC01” and “hLC02” humanized sequences represent novel amino acidsequences that comprise amino acid sequence changes in comparison toboth the original chimeric sequence and the partially humanizedsequences described herein.

The PTM mutations are intended to remove potentially detrimental posttranslational modification motifs from said proteins, whilst maintainingthe advantageous binding properties. The positions 1, 8, 9, 10, 13, 15,17, 19, 20, 21, 22, 30, 41, 43, 45, 49, 58, 63, 70, 77, 83, 85 and/or 87of hLC01 and hLC02 are preferably mutated (substituted) in comparison tothe original chimeric sequence.) The importance of the substitutionrelates primarily to the resulting amino acid, not the originating aminoacid. The change may therefore also be carried out from thecorresponding amino acid of the original chimeric amino acid or othervariant.

The following substitutions are novel over the chimeric and partiallyhumanized sequences:

-   -   the amino acid D1 of the LC (VL) sequence is substituted with E;    -   the amino acid V15 of the LC (VL) sequence is substituted with        P;    -   the amino acid D17 of the LC (VL) sequence is substituted with        E;    -   the amino acid V19 of the LC (VL) sequence is substituted with        A;    -   the amino acid T22 of the LC (VL) sequence is substituted with        S;    -   the amino acids D30 and S31 of the LC (VL) sequence is        substituted with any amino acid combination, preferably ES, SS,        TS, QS, HS, DH;    -   the amino acid V58 of the LC (VL) sequence is substituted with        I; and/or    -   the amino acid D70 of the LC (VL) sequence is substituted with        E.

Sequences that May be Modified in their CDR Binding Regions at ThoseResidues Required for Interaction with BCMA:

hLC03 - modified amino acids involved ininteraction with BCMA (SEQ ID NO 13): EIVMTQSPATLSVSPGERATLSCKASQSVDX ₁X ₂VX ₃ WX ₄QQKPGQAPRA LIX ₅ X ₆AX ₇ X ₈RX ₉ SGIPARFSGSX ₁₀ X₁₁GTEFTLTIISLQSEDFAVYY C X ₁₂QX ₁₃NNX ₁₄PX ₁₅TFGAGTKLELKR

Wherein Preferred Amino Acids are:

X₁: S, H, T, N, D, Q;

X₂: N, E, Q;

X₃: A, G, V, S, T, L, I;

X₄: Y, F, L, I, V, A, G;

X₅: Y, F, L;

X₆: S, T;

X₇: S, T, D, N, H, E, Q;

X₈: L, V, I, M;

X₉: F, L, I, V, Y, M;

X₁₀: G, X;

X₁₁: S, X;

X₁₂: Q, V, L, I, M;

X₁₃: Y, F, L, I, Q;

X₁₄: Y, F, R, Q, K; and/or

X₁₅: L, I, V, F.

The “hLC03 humanized sequence” represents novel amino acid sequencesthat comprise amino acid sequence changes in comparison to both theoriginal chimeric sequence and the partially humanized sequence. Thesesequence changes are intended to reflect potential changes in the aminoacids that bind the BCMA target, which may be substituted, whilstmaintaining the advantageous binding properties. The importance of thesubstitution relates primarily to the resulting amino acid, not theoriginating amino acid. The change may therefore also be carried outfrom the corresponding amino acid of the original chimeric amino acid orother variant.

For example:

-   -   the amino acid S31 of the LC (VL) sequence is S, H, T, N, D, Q;    -   the amino acid N32 of the LC (VL) sequence is N, E, Q;    -   the amino acid A34 of the LC (VL) sequence is A, G, V, S, T, L,        I;    -   the amino acid Y36 of the LC (VL) sequence is Y, F, L, I, V, A,        G;    -   the amino acid Y49 of the LC (VL) sequence is Y, F, L;    -   the amino acid S50 of the LC (VL) sequence is S, T;    -   the amino acid S52 of the LC (VL) sequence is S, T, D, N, H, E,        Q;    -   the amino acid L53 of the LC (VL) sequence is L, V, I, M;    -   the amino acid F55 of the LC (VL) sequence is F, L, I, V, Y, M;    -   the amino acid G66 of the LC (VL) sequence is G, X;    -   the amino acid S67 of the LC (VL) sequence is S, X;    -   the amino acid Q89 of the LC (VL) sequence is Q, V, L, I, M;    -   the amino acid Y91 of the LC (VL) sequence is Y, F, L, I, Q;    -   the amino acid Y94 of the LC (VL) sequence is Y, F, R, Q, K;        and/or    -   the amino acid L96 of the LC (VL) sequence is L, I, V, F.

In general, any change to a CDR region may also be considered as afeature of a CDR sequence when considered independently of the frameworksequence as a whole. Such modified CDR sequences may be considereddefining features of the present invention, either within or independentof their context in the entire framework region described herein. Forexample, the CDR sequences identified by underline in the hLC01 to hLC03may—in their unmodified or substituted form—be considered a definingfeature of the invention independently of the surrounding frameworksequence.

Example of Humanized LC Sequence:

hLC04 (SEQ ID NO 14): EIVMTQSPATLSVSPGERATLSCKASQSVESNVAWYQQKPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPL TFGAGTKLELKR

Alignments:

A CLUSTAL W (1.83) multiple sequence alignment of the variouspotentially amended sites within the LC sequence provides appropriatesequence comparisons in FIG. 9 . The “General sequence” represents an LCsequence, whereby each X represents a potential amino acid change.Preferred amino acid substitutions are those described above for each ofthe potentially mutated positions.

The present invention therefore relates to the humanized sequencesaccording to hHC01, hHC02, hHC03, hHC04, hHC05, hHC06, hHC07, hLC01,hLC02, hLC03 and/or hLC04, or any given combination thereof.

All possible combinations of potential modifications for any givenpotentially variant residue proposed herein (as identified by X in the“general” sequence) are encompassed by the present invention. Bycombining one or more of these various substitutions, humanized variantsmay be generated that exhibit the desired binding properties of thechimeric antibody originally developed and demonstrated herein. Theantibodies or parts thereof described herein also encompass a sequencewith at least 80%, preferably 90%, sequence identity to those humanizedsequences disclosed explicitly or disclosed through a sequence formula.

Preferred Embodiments Regarding the Antibody Epitope

The invention therefore relates to an isolated antibody or antibodyfragment that binds CD269 (BCMA), wherein the antibody binds an epitopecomprising one or more amino acids of residues 13 to 32 of CD269 (BCMA).

The amino acid sequence of residues 13 to 32 of CD269 are shown in SEQID No. 40. The N-terminus sequence of CD269 is provided in SEQ ID No.39. The extracellular domain of CD269 is provided as SEQ ID No. 38.

An antigen comprising the extracellular domain of CD269 according to SEQID No. 38 was used in vaccination in order to generate the bindingspecificity of the mouse and chimeric antibody described herein. Use ofthe entire CD269 protein, or fragments thereof comprising either amembrane-bound or intracellular domain, as an antigen during antibodygeneration could produce antibodies that bind concealed or intracellulardomains of CD269, thereby rendering such agents unsuitable ordisadvantageous for therapeutic application. The antibodies of thepresent invention are therefore defined by their binding to theextracellular portion of CD269. The specific epitope within theextracellular domain also represents a preferred novel and unexpectedcharacterising feature of the invention.

Fab fragments prepared from mouse or chimeric antibodies werecrystallized in complex with the purified BCMA extracellular domain andthe complex structure solved. The structural analysis has revealeddetailed information of the epitope of the antibody of the presentinvention and its biological relevance. The binding of an epitopecomprising one or more amino acids of residues 13 to 32 of CD269 (BCMA)of the extracellular domain by the antibody of the present invention isan advantageous property, as this region shows a significant overlapwith the binding sites of BAFF and APRIL, the two natural ligands ofCD269. No anti-CD269 antibody described in art to date has shown suchcomprehensive overlap with the BAFF and APRIL binding sites.

In one embodiment the isolated antibody or antibody fragment of thepresent invention is characterised in that the antibody binds an epitopecomprising one or more of amino acids 13, 15, 16, 17, 18, 19, 20, 22,23, 26, 27 or 32 of CD269 (BCMA). In another embodiment the isolatedantibody or antibody fragment of the present invention is characterisedin that the antibody binds an epitope consisting of amino acids 13, 15,16, 17, 18, 19, 20, 22, 23, 26, 27 and 32 of CD269 (BCMA). Theseresidues represent the amino acids that interact directly with theantibody of the present invention, as identified by the crystalstructure data provided herein. The numbering of these residues has beencarried out with respect to SEQ ID No. 39, which provides the N-terminalsequence of CD269.

In one embodiment of the invention the isolated antibody or antibodyfragment is characterised in that the antibody binding to CD269 (BCMA)disrupts the BAFF-CD269 and/or APRIL-CD269 interaction.

The binding of the antibodies of the present invention to theextracellular domain of CD269 disrupts the BAFF-CD269 interaction. Dueto the fact that the binding sites of APRIL and BAFF are positioned atsimilar sites to the antibody epitope, the binding of the antibody toCD269 will also block the APRIL-CD269 interaction.

Comparison of the specific epitope of the antibody of the presentinvention with the binding sites of APRIL and BAFF, for which crystalstructures have been solved and their interaction sites mapped, revealsa comprehensive overlap in the binding sites of the natural ligands andthe antibody as described herein. This represents a beneficial andunexpected aspect of the invention and enables a reliable and effectivedisruption of BAFF-CD269 and/or APRIL-CD269 interactions.

The invention therefore relates to an isolated antibody or antibodyfragment as described herein, wherein the antibody disrupts theAPRIL-CD269 interaction by binding an epitope comprising one or moreamino acids of residues 13, 15, 17, 18, 19, 22, 26, 27, 30, 31, 32, 33,34, 35 of CD269 (BCMA), in particular consisting of amino acids 13, 15,17, 18, 19, 22, 26, 27, 32. These amino acids correspond to the bindingsite of APRIL on CD269, and the overlapping residues of CD269 that bindboth the antibody as described herein and APRIL, respectively.

The invention therefore relates in another embodiment to an isolatedantibody or antibody fragment as described herein, wherein the antibodydisrupts the BAFF-CD269 interaction by binding an epitope comprising oneor more amino acids of residues 13, 15, 16, 17, 18, 19, 22, 25, 26, 27,29, 30, 31, 32, 34, 35 of CD269 (BCMA), in particular an epitopeconsisting of amino acids 13, 15, 16, 17, 18, 19, 22, 26, 27, 32. Theseamino acids correspond to the binding site of BAFF on CD269, and theoverlapping residues of CD269 that bind both the antibody as describedherein and BAFF, respectively.

Although antibodies that bind CD269 have been described in the art thatalso potentially disrupt APRIL- or BAFF-interactions with CD269, norelevant disclosure is provided relating to the specific epitope of suchantibodies. It cannot be assumed that the previously describedantibodies also bind an epitope with such a comprehensive overlap as theantibodies of the present invention. Even if APRIL- or BAFF-interactionswith CD269 have been shown to be disrupted, this could potentially occurdue to binding a considerably different epitope and subsequent sterichindrance of APRIL or BAFF docking. The degree of disruption of APRIL-or BAFF-interactions with CD269 caused by the antibodies of the priorhas not been documented previously.

The antibodies of the present invention enable an effective and reliabledisruption, which potentially represents an improved technical effect incomparison to those antibodies described in the art. An in vitroblocking assay can be performed for determination and comparison of BAFFand/or APRIL disruption, for example with the extracellular domain ofhuman BCMA and recombinant BAFF or APRIL.

In a preferred embodiment the epitope specificity, in combination withthe high affinity shown by the antibodies described herein, represents anovel and unexpected technical effect. In essence, the exceptionallyhigh affinity of the J22.9 antibody and the humanized variants thereof,provides not only “disruption” or “blocking” of the binding of thenatural ligands; but rather the ultra-high affinity of the antibodies ofthe invention ensures that the native ligands are essentially excludedcompletely or almost completely from binding their BCMA target when theantibody is present.

As disclosed in the examples below, the affinity of the humanizedantibodies as described herein is surprisingly high and comparativelybetter than similar approaches attempted in the prior art. A Kd in thepM range (as shown below) is commonly accepted as an outstandingaffinity not to be expected in common practice.

In another aspect the humanized antibody or antibody fragment of theinvention binds CD269 with high affinity, for example when measured bysurface plasmon resonance, such as Biacore, the antibody binds to humanCD269 with an affinity of 100 nM, 90, 80, 70, 60, 50, 40, 30 nM or less,or 20 nM or less, or an affinity of 15 nM or less, or an affinity of 5nM or less, or an affinity of 1000 pM or less, or an affinity of 500 pMor less, or an affinity of 100 pM or less, or 80 pM or less, or forexample about 50 pM.

In a further embodiment the antibody binds to human CD269 when measuredby surface plasmon resonance, such as Biacore, of between about 1 pM andabout 100 nM, or between about 100 pM and about 50 nM, or between about200 pM and about 20 nM.

Further Preferred Embodiments of the Invention

In one embodiment the invention relates to an antibody or antibodyfragment comprising an amino acid sequence defined by one or more of theamino acids that directly interact with the CD269 target and/or one ormore amino acids that interact via water interactions (see Tables 1 to6). The large number of water interactions involved in the binding ofthe antibody as described herein to the epitope represents an unusualand surprising aspect of the binding. In particular the high affinity ofthe antibody directed to the particular epitope described herein, incombination with the large number of water interactions involved in thebinding surface between the antibody and epitope, represents asurprising and unexpected aspect of the invention.

The invention therefore relates to an antibody or antibody fragmentcomprising an amino acid sequence as described herein, wherein thesequence is characterised by the presence of the specific amino acidresidues that are involved in the interaction surface with the targetepitope via a water bridge according to table 5, selected from the groupcomprising Ser31, Asn32, Tyr36, Ser50, Ser52, Gly66, Gln89, Tyr91 and/orTyr94 of the light chain, and/or Trp33, Ser35, Trp47, Glu50, Leu99and/or Tyr101 of the heavy chain, with respect to the chimera disclosedherein, or with respect to the corresponding residue of the humanizedsequence variants disclosed herein.

Although the examples with respect to water bridge formation of theantibody were carried out with the J22.9-xi chimera, the inventorsassert that this technical effect is maintained in the humanizedvariants of the present invention due to the maintenance of bindingcharacteristics in the humanized variants compared to the originalchimeric antibody tested. In the heavy chain, the only mutated waterbridge residue is Y101, but its water interaction involves a main chain(i.e. backbone) atom, and therefore can be reasonably assumed not tochange due to mutating the sidechain; in the light chain there are nomutations of residues involved in water bridges.

The antibody of the invention can be further characterised by the aminoacid residues of the epitope involved in the interaction via waterbridges with the antibody as described herein. The relevant features areprovided in Table 5. The invention is therefore, for example in oneembodiment, characterised in that residue Ser31 of the light chaininteracts with Thr32 of CD269 via a water molecule. Such a descriptionof the binding properties of the antibody of the present invention isintended for each interaction as provided in Table 5.

Furthermore, sequence variants of the antibodies described herein areencompassed in the present invention, in which one or more residuesinvolved in a “water bridging” interaction is modified in order to“substitute” a direct side-chain interaction into the sequence at theexpense of a water “bridge”. For example, a mutation or change could bemade in the amino acid sequence displacing the water from theinteraction interface but not substantially affecting the affinity ofthe interaction. The invention therefore relates to an antibody orantibody fragment comprising an amino acid sequence as described herein,wherein the sequence is characterised by sequence variation of thoseamino acid residues that are involved in the interaction surface withthe target epitope via a water bridge according to table 5, selectedfrom the group comprising Ser31, Ser31, Asn32, Tyr36, Ser50, Ser52,Gly66, Gln89, Tyr91 and/or Tyr94 of the light chain, and/or Trp33,Ser35, Trp47, Glu50, Leu99 and/or Tyr101 of the heavy chain, withrespect to the chimera disclosed herein, or with respect to thecorresponding residue of the humanized sequence variants disclosedherein. Variation at the corresponding positions of the humanizedantibodies described herein may relate to any given amino acidsubstitution, preferably an amino acid substitution that wouldeffectively displace the water from the interaction but maintain similarbinding properties with respect to epitope affinity and specificity.

In one embodiment of the invention the isolated antibody or antibodyfragment is characterised in that the antibody is glycosylated,preferably comprising an N-linked oligosaccharide chain, preferably atAsn297 of the heavy chain.

Glycosylation of the antibody refers to the attachment of carbohydratesor glycans to the antibody. N-linked glycans are attached to thenitrogen of asparagine or arginine side-chains. The carbohydrate chainsattached to the target proteins serve various functions. For instance,some proteins do not fold correctly unless they are glycosylated first.Also, polysaccharides linked at the amide nitrogen of asparagine in theprotein can confer stability on some secreted glycoproteins.Glycosylation in this case is not a strict requirement for properfolding, but unglycosylated protein can be degraded more quickly.

As is demonstrated in the examples of the present invention, thedeglycosylation of the antibody disclosed therein leads to a reductionin therapeutic effect in comparison to glycosylated forms of theantibody. It was surprising, that the glycosylation would play asignificant role in maintaining activity of the antibody. Theglycosylation therefore represents a preferred embodiment of theinvention associated with unexpected technical advantages.

As demonstrated in the examples herein, although the overall tumor loadof animals treated with J22.9-xi-N-glycan (deglycosylated) was notsignificantly different from animals receiving the isotype controlantibody, the lifespan of these mice was substantially increasedcompared to the isoAb-treated group. Since J22.9-xi-N-glycan was shownto be unable to induce ADCC or CDC, this result indicates that alone thebinding of J22.9-xi to BCMA hinders tumor growth. It may be reasonablyconsidered that this is due to blocking of the interaction between thereceptor and its native ligands (APRIL and BAFF). This aspect of theinvention and the antibodies described herein represents a surprisingtechnical effect, which could not have been derived from the antibodiesof the prior art. The J22.9-xi-N-glycan (deglycosylated) can beconsidered a control sample in the experiments described which enablesthe binding of the antibody to its target epitope, without thedownstream effects of ADCC or CDC, to be assessed for potentiallytherapeutic effect. The antibodies of the invention, preferably withglycosylation, therefore demonstrate such an effective epitope bindingthat enables the prevention (or significant disruption) of binding bythe natural ligands to lead to cell toxicity. This characteristic of theantibodies described herein has not been described for similarantibodies described in the art.

Although the examples with respect to glycosylation of the antibody werecarried out with the J22.9-xi chimera, the inventors assert that thistechnical effect is maintained in the humanized variants of the presentinvention due to the maintenance of binding characteristics in thehumanized variants compared to the original chimeric antibody tested.The preferred position of glycosylation (Asn297 of the heavy chain) hasno direct connection to any of the mutated residues and lies in thehuman constant region of the full IgG. It is therefore reasonable toassume that no differences in the glycosylation pattern at this positionexist in any of the J22.9 variants compared to the chimeric antibody.

Use and Functional Aspects of the Invention

The antibodies of the present invention are capable of binding theepitopes described herein, blocking interaction of the natural ligandsof this epitope, and inducing CDC and ADCC.

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) is one factorinduced by the antibody of the present invention that generates thedesired therapeutic effect. ADCC is a mechanism of cell-mediated immunedefense, whereby an effector cell of the immune system actively lyses atarget cell, whose membrane-surface antigens have been bound by specificantibodies. After binding of CD269-expressing cells by the antibodies ofthe present invention ADCC may be induced. Classical ADCC is mediated bynatural killer (NK) cells; macrophages, neutrophils and eosinophils canalso mediate ADCC. ADCC is part of the adaptive immune response due toits dependence on a prior antibody response. Experiments in mice mayindicate that ADCC is an important mechanism of action of therapeuticantibodies as described herein.

A preferred embodiment of the invention relates to the isolated antibodyor antibody fragment as described herein for use as a medicament in thetreatment of a medical disorder associated with the presence ofpathogenic B cells.

In one embodiment of the invention the medical disorder is aCD269-associated disorder, preferably associated with pathogenic Bcells, which is preferably a disease of plasma cells and/or memory Bcells.

A disease of plasma cells may be a cancer of plasma cells, for examplemultiple myeloma, plasmacytoma, WaldenstrOm macroglobulinemia or plasmacell leukemia. A disease of plasma cells may be a cancer of Blymphocytes, such as Hodgkin's disease.

In one embodiment of the invention the medical disorder is an autoimmunedisease associated with autoreactive plasma cells and/or autoreactivememory B cells, such as an inflammatory autoimmune disease, for examplesystemic lupus erythematosus or rheumatic arthritis.

The invention therefore also encompasses a method of treatment for themedical disorders as disclosed herein, preferably comprising theadministration of a therapeutically effective amount of antibody to asubject in need of such treatment.

A further aspect of the invention relates to an antibody-drug conjugate(ADC) comprising the antibody or antibody fragment as described herein.Anti-CD269 Antibody-Drug Conjugates “anti-CD269 ADC” can be described asan anti-CD269 antibody or fragment thereof conjugated to a therapeuticagent. In certain embodiments, the ADC comprises an anti-CD269 antibody(e.g., a humanized variant of J22.9-xi as described herein).

The ADCs or ADC derivatives as described herein produce clinicallybeneficial effects on CD269-expressing cells when administered to asubject with a CD269-expressing medical condition, such as cancer orautoimmune disorder. In one embodiment, the anti-CD269 antibody orderivative thereof is conjugated to a cytotoxic agent, such that theresulting ADC or ADC derivative exerts a cytotoxic effect on aCD269-expressing cancer cell, preferably when taken up or internalizedby the cell.

The anti-CD269 ADC or ADC derivative is preferably internalized andaccumulates within a CD269-expressing cell, where the ADC or ADCderivative exerts a therapeutic effect (e.g., a cytotoxic effect).Particularly suitable moieties for conjugation to antibodies or antibodyderivatives are chemotherapeutic agents, prodrug converting enzymes,radioactive isotopes or compounds, or toxins. For example, an anti-CD269antibody or derivative thereof can be conjugated to a cytotoxic agentsuch as a chemotherapeutic agent, or a toxin (e.g., a cytostatic orcytocidal agent).

Another aspect of the invention relates to a preferably isolated nucleicacid molecule selected from the group consisting of:

-   -   a) a nucleic acid molecule comprising a nucleotide sequence        -   which encodes an isolated antibody or antibody fragment            according to any one of the preceding claims,        -   which encodes an amino acid sequence selected from the group            consisting of those sequences according to SEQ ID 1 to 31            and 41 to 42,        -   comprising a sequence or sequence fragment of SEQ ID No. 32            to 36,    -   b) a nucleic acid molecule which is complementary to a        nucleotide sequence in accordance with a);    -   c) a nucleic acid molecule comprising a nucleotide sequence        having sufficient sequence identity to be functionally        analogous/equivalent to a nucleotide sequence according to a) or        b), comprising preferably a sequence identity to a nucleotide        sequence according to a) or b) of at least 80%, preferably 90%,        more preferably 95%;    -   d) a nucleic acid molecule which, as a consequence of the        genetic code, is degenerated into a nucleotide sequence        according to a) through c); and    -   e) a nucleic acid molecule according to a nucleotide sequence        of a) through d) which is modified by deletions, additions,        substitutions, translocations, inversions and/or insertions and        functionally analogous/equivalent to a nucleotide sequence        according to a) through d).

A further aspect of the invention relates to a host cell, such as abacterial cell or mammalian cell, preferably a hybridoma cell or cellline, capable of producing an antibody or antibody fragment as describedherein, and/or comprising a nucleic acid molecule as described herein.

A further aspect of the invention relates to a pharmaceuticalcomposition comprising the isolated antibody or antibody fragment asdescribed herein, a nucleic acid molecule as described herein or a hostcell as described herein, together with a pharmaceutically acceptablecarrier.

An additional and surprising aspect of the invention is an improvedstability of the antibody as disclosed herein. The antibody can readilybe stored for extended periods under appropriate conditions without anyloss of binding affinity. Appropriate tests have been carried outregarding maintenance of activity after storage at either −80 or 4 degC., which demonstrate unexpectedly good stability of the antibody andmaintenance of activity after storage at both aforementionedtemperatures (FIG. 3 c ). This improved stability is evident for thechimeric antibody, and surprisingly also for the humanized variantsthereof. Unexpectedly the stability of the humanized variants isimproved over the chimera under long term storage.

A further advantage of the antibodies as described herein the effectivesystemic depletion of myeloma cells as demonstrated in the examples.Antibodies previously disclosed in the prior art have not beendemonstrated to exhibit the desired anti-plasma cell effect in asystemic manner. Studies carried out on the antibodies of the prior artdisclose only sub-cutaneous injection with myeloma cells and subsequenttreatment of the isolated cell mass. The present invention provides anantibody capable of systemic depletion of cancerous multiple myelomacells after their i.v. injection, as demonstrated in the examples. Theeffective depletion of targeted cells represents a technical effect thathas not previously been demonstrated in the prior art in addition to abeneficial property of the antibodies of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is demonstrated by way of the example by the examples andfigures disclosed herein. The figures provided herein representparticular embodiments of the invention and are not intended to limitthe scope of the invention. The figures are to be considered asproviding a further description of possible and potentially preferredembodiments that enhance the technical support of one or morenon-limiting embodiments.

FIG. 1 : In vitro characterization of J22.9-xi. Concentration dependentbinding of J22.9-xi to BCMA in (a) ELISA and (b) by flow cytometry usingCD269-positive MM.1S cells. (c) Binding affinity of J22.9-xi to BCMA wasdetermined from surface plasmon resonance measurements with theindicated concentrations of BCMA. (d) J22.9-xi blocks the interactionbetween BAFF and BCMA adsorbed onto microtiter plates.

FIG. 2 : The structure of CD269 (BCMA) and the J22.9-xi Fab:CD269complex. (a) CD269 (BCMA) recognition surface. Three views of theextracellular domain of CD269 (BCMA) showing the binding epitoperesidues for BAFF/APRIL and J22.9-xi. At top, a view directly on thebinding face of CD269 (BCMA): the light grey shading indicates allresidues comprising the binding epitope of BAFF and APRIL as identifiedfrom their crystal structures, black residues (shown as spheres) do notcontact either BAFF, APRIL or J22.9-xi; the subset of epitope residuesinvolved in J22.9-xi binding are shown in surface representation;remaining light grey residues shown as spheres are part of both the BAFFand APRIL epitopes but make no direct contacts to J22.9-xi. The middleand lower panels show the same representation as in the top panel butrotated 90° toward and away from the viewer, respectively. (b) Two viewsof the J22.9-xi Fab:CD269 complex. J22.9-xi is shown in surfacerepresentation with the heavy chain coloured light grey and the lightchain in dark grey. CD269 (BCMA) is shown in ribbon representation boundto the J22.9-xi antigen pocket. At left, full view of the Fab:CD269complex; at right, the complex tilted toward the viewer to show thebinding pocket.

FIG. 3 : In vitro cytotoxicity of J22.9-xi. (a) CD269-positive MM.1S-Luccells mixed with human PBMCs at an effector to target ratio of 20:1 wereincubated with the indicated concentrations of J22.9-xi for 4 hours.Open symbols indicate cytotoxic activity of J22.9-xi without —N-glycanswhen incubated with PBMCs from donor 1 and 2. Error bars indicate SEM.(b) Deglycosylation does not affect J22.9-xi binding to MM.1S cells. (c)Storage of J22.9-xi for 3 weeks at 4° C. or −80° C. does not affectcytotoxicity.

FIG. 4 : Efficacy of J22.9-xi in xenografted NSG mice. (a) Tumordevelopment over time with administration of 200 μg of J22.9-xi or thecontrol antibody twice a week, and untreated control mice. (b) Totaltumor burden between day 6 and 41 (Area under the curve (AUC) of (a)).Plotted are the mean values with SEM (**P<0.01, ***P<0.001, t-test). (c)Overall survival of J22.9-xi and isotype control mice. The P value wascalculated using the Log-rank (Mantel-Cox) Test. (d-1) Detection ofMM.1S-Luc cells in the indicated groups without administration oftherapeutic antibody. Below the rightmost image the numbers (41, 41, 44,40) indicate the days post-tumor cell injection on which the specificmouse died. (d-2) Detection of MM.1 S-Luc cells in the indicated groupsat day 21 and 28. Dorsal view. (e) Relationship between J22.9-xiconcentration and tumor development. (f) Total tumor load between day 6and 42 (AUC of (e)). Mean values with SEM (**P<0.01, ***P<0.001,t-test). (g) Overview of experimental time line.

FIG. 5 : Treatment of established tumors. (a) Tumor development overtime with administration of 200 μg of J22.9-xi or control antibody twiceweekly, and untreated control mice. (b) Total tumor load between day 8and 48 (AUC of (a)). Plotted are the mean values with SEM (*P<0.05,**P<0.01, t-test). (c) Overall survival of J22.9-xi and isotype controlmice. The P value was calculated using the Log-rank (Mantel-Cox) Test.(d) An overview of the experimental time line is provided.

FIG. 6 : Tumor treatment in the early phase of disease. (a) Course oftumor growth when treated with 2 μg, 20 μg or 200 μg of J22.9-xi or 200μg of either the isotype control antibody or J22.9-xi without—N-glycans, and without tumor. (b) Total tumor burden within day 9 to 44(AUC of (a)). Shown are the mean values with SEM (*P<0.05, **P<0.01,t-test). (c) Survival of antibody-treated and control xenograftSCID-Beige mice. The P values were calculated using Log-rank(Mantel-Cox) Test. (d) An overview of the experimental time line isprovided.

FIG. 7 : Instability of hybridoma J22.9. The supernatant of hybridomaJ22.9 tested positive for binding to BCMA in ELISA on BCMA-coatedmicrotiter plates at day 1. Later analysis at indicated time pointsrevealed a reduction of binding capacity of the supernatant. The mediumwas exchanged on days 7, 14 and 21.

FIG. 8 : Summary of the sequences of the humanized antibodies comparedto J22.9-xi. Sequence comparisons were carried out using standardalignment software. HCg: SEQ ID NO: 41; HCm: SEQ ID NO: 1; HCpH: SEQ IDNO: 2; hHC01: SEQ ID NO: 3; hHC02: SEQ ID NO: 4; hHC03: SEQ ID NO: 5;hHC04: SEQ ID NO: 6; hHC05: SEQ ID NO: 7; hHC06: SEQ ID NO: 8; andhHC07: SEQ ID NO: 9.

FIG. 9 : Summary of the sequences of the humanized antibodies comparedto J22.9-xi. Sequence comparisons were carried out using standardalignment software. LCg: SEQ ID NO: 42; LCm: SEQ ID NO: 43; LCpH: SEQ IDNO: 10; hLC01: SEQ ID NO: 11; hLC02: SEQ ID NO: 12; hLC03: SEQ ID NO:13; and hLC04: SEQ ID NO: 14.

FIG. 10 : Sequence optimized variants of J22.9-xi show similar bindingin ELISA. Binding of the chimeric J22.9-xi and humanized variants wastested via ELISA using human BCMA (hBCMA) or cynomolgous BCMA (cyBCMA)coated microtiter plates (J22.9-H corresponds to humanized sequence SEQID No. 27; J22.9-FSY corresponds to humanized and PTM modified SEQ IDNo. 28; J22.9-ISY corresponds to humanized and PTM modified SEQ ID No.29).

FIG. 11 : Sequence optimized variants of J22.9-xi show similar bindingin flow cytometry. Binding of the chimeric J22.9-xi and humanizedvariants was tested via flow cytometry using the human MM cell lineRPMI-8226 (J22.9-FSY corresponds to humanized and PTM modified SEQ IDNo. 28; J22.9-ISY corresponds to humanized and PTM modified SEQ ID No.29).

FIG. 12 : SPR raw data: Binding affinities of the chimeric J22.9-xi andhumanized variants to human (A) and cynomolgus (B) BCMA was measured bySurface Plasmon Resonance (SPR) Spectrometry. IgGs were immobilized viaamine chemistry to a Proteon GLH sensor chip and binding measured withBCMA in the mobile phase. The order of raw data traces in the graphcorresponds to the order to samples listed in the legend of the figure.

FIG. 13 : Gel electrophoresis of antibody variants. Antibody variantswere run in non-reduced SDS-PAGE and stained to show protein migration.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, an “antibody” generally refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes or fragments of immunoglobulin genes. Where the term “antibody” isused, the term “antibody fragment” may also be considered to be referredto. The recognized immunoglobulin genes include the kappa, lambda,alpha, gamma, delta, epsilon and mu constant region genes, as well asthe myriad immunoglobulin variable region genes. Light chains areclassified as either kappa or lambda. Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Thebasic immunoglobulin (antibody) structural unit is known to comprise atetramer or dimer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (L) (about 25 kD) andone “heavy” (H) chain (about 50-70 kD). The N-terminus of each chaindefines a variable region of about 100 to 110 or more amino acids,primarily responsible for antigen recognition. The terms “variable lightchain” and “variable heavy chain” refer to these variable regions of thelight and heavy chains respectively. Optionally, the antibody or theimmunological portion of the antibody, can be chemically conjugated to,or expressed as, a fusion protein with other proteins.

The antibodies of the invention are intended to bind against mammalian,in particular human, protein targets. The use of protein names maycorrespond to either mouse or human versions of a protein.

“Specific binding” is to be understood as via one skilled in the art,whereby the skilled person is clearly aware of various experimentalprocedures that can be used to test binding and binding specificity.Some cross-reaction or background binding may be inevitable in manyprotein-protein interactions; this is not to detract from the“specificity” of the binding between antibody and epitope. The term“directed against” is also applicable when considering the term“specificity” in understanding the interaction between antibody andepitope.

Antibodies of the invention include, but are not limited to polyclonal,monoclonal, bispecific, human, humanized or chimeric antibodies, singlevariable fragments (ssFv), single domain antibodies (such as VHHfragments from nanobodies), single chain fragments (scFv), Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic antibodies and epitope-binding fragments orcombinations thereof of any of the above, provided that they retain theoriginal binding properties. Also mini-antibodies and multivalentantibodies such as diabodies, triabodies, tetravalent antibodies andpeptabodies can be used in a method of the invention. The immunoglobulinmolecules of the invention can be of any class (i.e. IgG, IgE, IgM, IgDand IgA) or subclass of immunoglobulin molecules. Thus, the termantibody, as used herein, also includes antibodies and antibodyfragments either produced by the modification of whole antibodies orsynthesized de novo using recombinant DNA methodologies.

Humanized antibody comprising one or more CDRs of antibodies of theinvention or one or more CDRs derived from said antibodies can be madeusing any methods known in the art. For example, four general steps maybe used to humanize a monoclonal antibody. These are: (1) determiningthe nucleotide and predicted amino acid sequence of the startingantibody light and heavy variable domains (2) designing the humanizedantibody, i.e., deciding which antibody framework region to use duringthe humanizing process (3) the actual humanizingmethodologies/techniques and (4) the transfection and expression of thehumanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;5,585,089; 6,180,370; 5,225,539; 6,548,640.

The term humanized antibody means that at least a portion of theframework regions, and optionally a portion of CDR regions or otherregions involved in binding, of an immunoglobulin is derived from oradjusted to human immunoglobulin sequences. The humanized, chimeric orpartially humanized versions of the mouse monoclonal antibodies can, forexample, be made by means of recombinant DNA technology, departing fromthe mouse and/or human genomic DNA sequences coding for H and L chainsor from cDNA clones coding for H and L chains. Humanized forms of mouseantibodies can be generated by linking the CDR regions of non-humanantibodies to human constant regions by recombinant DNA techniques(Queen et al., 1989; WO 90/07861). Alternatively the monoclonalantibodies used in the method of the invention may be human monoclonalantibodies. Human antibodies can be obtained, for example, usingphage-display methods (WO 91/17271; WO 92/01047).

As used herein, humanized antibodies refer also to forms of non-human(e.g. murine, camel, llama, shark) antibodies that are specific chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin.

As used herein, human or humanized antibody means an antibody having anamino acid sequence corresponding to that of an antibody produced by ahuman and/or has been made using any of the techniques for making humanantibodies known in the art or disclosed herein. Human antibodies can beselected by competitive binding experiments, or otherwise, to have thesame epitope specificity as a particular mouse antibody. The humanizedantibodies of the present invention surprisingly share the usefulfunctional properties of the mouse antibodies to a large extent. Humanpolyclonal antibodies can also be provided in the form of serum fromhumans immunized with an immunogenic agent. Optionally, such polyclonalantibodies can be concentrated by affinity purification using amyloidfibrillar and/or non-fibrillar polypeptides or fragments thereof as anaffinity reagent. Monoclonal antibodies can be obtained from serumaccording to the technique described in WO 99/60846.

The present invention further relates to the use of the antibodies, orfragments thereof, as described herein, for example the variableregions, in recognition molecules or affinity reagents that are suitablefor selective binding to a target. The affinity reagent, antibody orfragment thereof according to the invention may be PEGylated, wherebyPEGylation refers to covalent attachment of polyethylene glycol (PEG)polymer chains to the inventive antibody. PEGylation may be routinelyachieved by incubation of a reactive derivative of PEG with the targetmolecule. PEGylation to the antibody can potentially mask the agent fromthe hosts immune system, leading to reduced immunogenicity andantigenicity or increase the hydrodynamic size of the agent which mayprolong its circulatory time by reducing renal clearance.

A variable region of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al. (1997) J.Molec. Biol. 273:927-948). As used herein, a CDR may refer to CDRsdefined by either approach or by a combination of both approaches.

In some embodiments, the invention provides an antibody, which comprisesat least one CDR, at least two, at least three, or more CDRs that aresubstantially identical to at least one CDR, at least two, at leastthree, or more CDRs of the antibody of the invention. Other embodimentsinclude antibodies which have at least two, three, four, five, or sixCDR(s) that are substantially identical to at least two, three, four,five or six CDRs of the antibodies of the invention or derived from theantibodies of the invention. In some embodiments, the at least one, two,three, four, five, or six CDR(s) are at least about 85%, 86%, 87%, 88%,89%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one, two orthree CDRs of the antibody of the invention. It is understood that, forpurposes of this invention, binding specificity and/or overall activityis generally retained, although the extent of activity may vary comparedto said antibody (may be greater or lesser).

The half life and cytotoxic potential of an antibody are dependentprimarily on the interaction of the Fc-domain with differentFc-gamma-receptors. In the case of the antibody half life, the neonatalFc receptor (FcRn) plays a major role. This receptor is expressed onseveral cell types and tissues such as monocytes and vascular endotheliacells that are able to take up serum proteins into their recyclingendosomes. In the endosomes, the pH is decreased to approximately 6 andunder these conditions the antibodies are able to bind to FcRn. Thisinteraction protects the antibodies from degradation until they areagain released into the blood where the physiological pH disrupts thebinding to the receptor (Roopenian and Akilesh (2007) Nat Rev Immunol7:715-725). The higher the affinity of the antibody to the FcRn at pH 6,the greater the half life of that antibody. Fc-fragment mutations knownto stabilize this interaction are summarised in Presta (2008, Curr OpinImmunol 20:460-470).

Therapeutic antibodies can act through several mechanisms upon bindingto their target. The binding itself can trigger signal transduction,which can lead to programmed cell death (Chavez-Galan et al. (2009) CellMol Immunol 6:15-25). It can also block the interaction of a receptorwith its ligand by either binding to the receptor or the ligand. Thisinterruption can cause apoptosis if signals important for survival areaffected (Chiu et al. (2007) Blood 109:729-739). With regard tocell-depletion there are two major effector mechanisms known. The firstis the complement-dependent cytotoxicity (CDC) towards the target cell.There are three different pathways known. However, in the case ofantibodies the important pathway for CDC is the classical pathway whichis initiated through the binding of Clq to the constant region of IgG orIgM (Wang and Weiner (2008) Expert Opin Biol Ther 8:759-768).

The second mechanism is called antibody-dependent cellular cytotoxicity(ADCC). This effector function is characterized by the recruitment ofimmune cells which express Fc-receptors for the respective isotype ofthe antibody. ADCC is largely mediated by activating Fc-gamma receptors(FcγR) which are able to bind to IgG molecules either alone or as immunecomplexes. Mice exhibit three (FcγRI, FcγRIII and FcγRIV) and humansfive (FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA and FcγRIIIB) activatingFcγ-receptors. These receptors are expressed on innate immune cells likegranulocytes, monocytes, macrophages, dendritic cells and natural killercells and therefore link the innate with the adaptive immune system.Depending on the cell type there are several modes of action ofFcgR-bearing cells upon recognition of an antibody-marked target cell.Granulocytes generally release vasoactive and cytotoxic substances orchemoattractants but are also capable of phagocytosis. Monocytes andmacrophages respond with phagocytosis, oxidative burst, cytotoxicity orthe release of pro-inflammatory cytokines whereas Natural killer cellsrelease granzymes and perforin and can also trigger cell death throughthe interaction with FAS on the target cell and their Fas ligand(Nimmerjahn and Ravetch (2008) Nat Rev Immunol 8:34-47; Wang and Weiner(2008) Expert Opin Biol Ther 8:759-768; Chavez-Galan et al. (2009) CellMol Immunol 6:15-25).

The antibody-dependent cellular cytotoxicity (ADCC) can also be improvedby strengthening the binding of the Fc-domain to activating Fc-gammareceptors (FcγR). This can also be achieved through mutations in theFc-gamma domain as summarized in Presta (2008, Curr Opin Immunol20:460-470).

Another way to change the ADCC is manipulation of the sugar moietypresent on each IgG at Asn297. Defucolylation and removal of sialic acidfrom the end of the sugar molecules are known to increase the cytotoxicpotential of an antibody (Anthony and Ravetch (2010) J Clin Immunol 30Suppl 1:S9-14).

Sequence variants of the claimed nucleic acids, proteins and antibodies,for example defined by the claimed % sequence identity, that maintainthe said properties of the invention are also included in the scope ofthe invention. Such variants, which show alternative sequences, butmaintain essentially the same binding properties, such as targetspecificity, as the specific sequences provided are known as functionalanalogues, or as functionally analogous. Sequence identity relates tothe percentage of identical nucleotides or amino acids when carrying outa sequence alignment.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology or sequence identity to thenucleotide sequence of any native gene. Nonetheless, polynucleotidesthat vary due to differences in codon usage are specificallycontemplated by the present invention. Deletions, substitutions andother changes in sequence that fall under the described sequenceidentity are also encompassed in the invention.

Protein sequence modifications, which may occur through substitutions,are also included within the scope of the invention. Substitutions asdefined herein are modifications made to the amino acid sequence of theprotein, whereby one or more amino acids are replaced with the samenumber of (different) amino acids, producing a protein which contains adifferent amino acid sequence than the primary protein, preferablywithout significantly altering the function of the protein. Likeadditions, substitutions may be natural or artificial. It is well knownin the art that amino acid substitutions may be made withoutsignificantly altering the protein's function. This is particularly truewhen the modification relates to a “conservative” amino acidsubstitution, which is the substitution of one amino acid for another ofsimilar properties. Such “conserved” amino acids can be natural orsynthetic amino acids which because of size, charge, polarity andconformation can be substituted without significantly affecting thestructure and function of the protein. Frequently, many amino acids maybe substituted by conservative amino acids without deleteriouslyaffecting the protein's function.

In general, the non-polar amino acids Gly, Ala, Val, Ile and Leu; thenon-polar aromatic amino acids Phe, Trp and Tyr; the neutral polar aminoacids Ser, Thr, Cys, Gln, Asn and Met; the positively charged aminoacids Lys, Arg and His; the negatively charged amino acids Asp and Glu,represent groups of conservative amino acids. This list is notexhaustive. For example, it is well known that Ala, Gly, Ser andsometimes Cys can substitute for each other even though they belong todifferent groups.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in the tableimmediately below, or as further described below in reference to aminoacid classes, may be introduced and the products screened.

Potential Amino Acid Substitutions:

Preferred Original conservative Examples of residue substitutionsexemplary substitutions Ala (A) Val Val; Leu; Ile Asg (R) Lys Lys; Gln;Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) SerSer; Ala Gln (Q) Asn Asn, Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His(H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe;Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys (K) ArgArg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala;Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; PheTyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala;Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.

Conservative amino acid substitutions are not limited to naturallyoccurring amino acids, but also include synthetic amino acids. Commonlyused synthetic amino acids are omega amino acids of various chainlengths and cyclohexyl alanine which are neutral non-polar analogs;citrulline and methionine sulfoxide which are neutral non-polar analogs,phenylglycine which is an aromatic neutral analog; cysteic acid which isa negatively charged analog and ornithine which is a positively chargedamino acid analog. Like the naturally occurring amino acids, this listis not exhaustive, but merely exemplary of the substitutions that arewell known in the art.

The antibodies of the present invention may be produced by transfectionof a host cell with an expression vector comprising the coding sequencefor the antibody of the invention. An expression vector or recombinantplasmid is produced by placing these coding sequences for the antibodyin operative association with conventional regulatory control sequencescapable of controlling the replication and expression in, and/orsecretion from, a host cell. Regulatory sequences include promotersequences, e.g., CMV promoter, and signal sequences which can be derivedfrom other known antibodies. Similarly, a second expression vector canbe produced having a DNA sequence which encodes a complementary antibodylight or heavy chain. In certain embodiments this second expressionvector is identical to the first except insofar as the coding sequencesand selectable markers are concerned, so to ensure as far as possiblethat each polypeptide chain is functionally expressed. Alternatively,the heavy and light chain coding sequences for the antibody may resideon a single vector.

A selected host cell is co-transfected by conventional techniques withboth the first and second vectors (or simply transfected by a singlevector) to create the transfected host cell of the invention comprisingboth the recombinant or synthetic light and heavy chains. Thetransfected cell is then cultured by conventional techniques to producethe engineered antibody of the invention. The antibody which includesthe association of both the recombinant heavy chain and/or light chainis screened from culture by appropriate assay, such as ELISA or RIA.Similar conventional techniques may be employed to construct otherantibodies.

Suitable vectors for the cloning and subcloning steps employed in themethods and construction of the compositions of this invention may beselected by one of skill in the art. For example, the conventional pUCseries of cloning vectors may be used. One vector, pUC19, iscommercially available. The components of such vectors, e.g. replicons,selection genes, enhancers, promoters, signal sequences and the like,may be obtained from commercial or natural sources or synthesized byknown procedures for use in directing the expression and/or secretion ofthe product of the recombinant DNA in a selected host. Other appropriateexpression vectors of which numerous types are known in the art formammalian, bacterial, insect, yeast, and fungal expression may also beselected for this purpose.

The present invention also encompasses a cell line transfected with arecombinant plasmid containing the coding sequences of the antibodies ofthe present invention. Host cells useful for the cloning and othermanipulations of these cloning vectors are also conventional.

Suitable host cells or cell lines for the expression of the antibodiesof the invention include mammalian cells such as NSO, Sp2/0, CHO (e.g.DG44), COS, HEK, a fibroblast cell (e.g., 3T3), and myeloma cells, forexample it may be expressed in a CHO or a myeloma cell. Human cells maybe used, thus enabling the molecule to be modified with humanglycosylation patterns. Alternatively, other prokaryotic or eukaryoticcell lines may be employed. The selection of suitable mammalian hostcells and methods for transformation, culture, amplification, screeningand product production and purification are known in the art.

In accordance with the present invention there is provided a method ofproducing an anti-CD269-antibody of the present invention which binds toand neutralises the activity of human CD269 which method comprises thesteps of; providing a first vector encoding a heavy chain of theantibody; providing a second vector encoding a light chain of theantibody; transforming a mammalian host cell (e.g. CHO) with said firstand second vectors; culturing the host cell of step (c) under conditionsconducive to the secretion of the antibody from said host cell into saidculture media; recovering the secreted antibody of step (d). Onceexpressed, the antibody can be assessed for the desired bindingproperties using methods as described herein.

The invention encompasses immunoconjugates (interchangeably referred toas “antibody-drug conjugates” or “ADCs”) comprising an antibodyaccording to the invention as herein described including, but notlimited to, an antibody conjugated to one or more cytotoxic agents, suchas a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin(e.g., a protein toxin, an enzymatically active toxin of bacterial,fungal, plant, or animal origin, or fragments thereof), or a radioactiveisotope (i.e., a radioconjugate). Techniques for conjugating therapeuticagents to proteins, and in particular to antibodies, such as for theAnti-CD269 Antibody-Drug Conjugates of the present invention, arewell-known. (See, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy,” in Monoclonal AntibodiesAnd Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985);Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled DrugDelivery (Robinson et al. eds., Marcel Dekker, Inc., 2nd ed. 1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview,” in Monoclonal Antibodies '84: Biological And ClinicalApplications (Pinchera et al. eds., 1985); “Analysis, Results, andFuture Prospective of the Therapeutic Use of Radiolabeled Antibody InCancer Therapy,” in Monoclonal Antibodies For Cancer Detection AndTherapy (Baldwin et al. eds., Academic Press, 1995); and Thorpe et al.,1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO89/12624.)

Typically, the ADC or ADC derivative comprises a linker region betweenthe therapeutic agent and the anti-CD269 antibody or derivative thereof.As noted supra, in typical embodiments, the linker is cleavable underintracellular conditions, such that cleavage of the linker releases thetherapeutic agent from the antibody in the intracellular environment.For example, in some embodiments, the linker is cleavable by a cleavingagent that is present in the intracellular environment (e.g., within alysosome or endosome or caveolae). The linker can be, e.g., a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Inother embodiments, the cleavable linker is pH-sensitive, i.e., sensitiveto hydrolysis at certain pH values. Typically, the pH-sensitive linkeris hydrolyzable under acidic conditions. In yet other embodiments, thelinker is cleavable under reducing conditions (e.g., a disulfidelinker). A variety of disulfide linkers are known in the art (See forexample Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates inRadioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press,1987. See also U.S. Pat. No. 4,880,935.)

Typically, the linker is not substantially sensitive to theextracellular environment. In other, non-mutually exclusive embodiments,the linker promotes cellular internalization. In certain embodiments,the linker promotes cellular internalization when conjugated to thetherapeutic agent (i.e., in the milieu of the linker-therapeutic agentmoiety of the ADC or ADC derivate as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the therapeutic agent and the anti-CD269 antibody orderivative thereof (i.e., in the milieu of the ADC or ADC derivative asdescribed herein). A variety of linkers that can be used with thepresent compositions and methods are described in WO 2004010957 entitled“Drug Conjugates and Their Use for Treating Cancer, An AutoimmuneDisease or an Infectious Disease” filed Jul. 31, 2003, and U.S.Provisional Application No. 60/400,403, entitled “Drug Conjugates andtheir use for treating cancer, an autoimmune disease or an infectiousdisease”, filed Jul. 31, 2002 (the disclosure of which is incorporatedby reference herein).

In certain embodiments, an immunoconjugate comprises an antibody asdescribed herein, including but not limited to, an antibody and achemotherapeutic agent or other toxin. Enzymatically active toxins andfragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies.

Antibodies or fragments thereof of the present invention may also beconjugated to one or more toxins, including, but not limited to, acalicheamicin, maytansinoids, dolastatins, aurostatins, a trichothecene,and CC1065, and the derivatives of these toxins that have toxinactivity. Suitable cytotoxic agents include, but are not limited to, anauristatin includingdovaline-valine-dolaisoleunine-dolaproine-phenylalanine (MMAF) andmonomethyl auristatin E (MMAE) as well as ester forms of MMAE, a DNAminor groove binding agent, a DNA minor groove alkylating agent, anenediyne, a lexitropsin, a duocarmycin, a taxane, including paclitaxeland docetaxel, a puromycin, a dolastatin, a maytansinoid, and a vincaalkaloid. Specific cytotoxic agents include topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine,DM-1, DM-4, netropsin. Other suitable cytotoxic agents includeanti-tubulin agents, such as an auristatin, a vinca alkaloid, apodophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, amaytansinoid, a combretastatin, or a dolastatin. Antitubulin agentincludedimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylened-iamine(AFP), MMAF, MMAE, auristatin E, vincristine, vinblastine, vindesine,vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A,epothilone B, nocodazole, colchicines, colcimid, estramustine,cemadotin, discodermolide, maytansine, DM-1, DM-4 or eleutherobin.

In some embodiments, the immunoconjugate comprises an antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin (which are pentapeptide derivatives ofdolastatins) drug moiety may be attached to the antibody through the N(amino) terminus or the C (carboxyl) terminus of the peptidic drugmoiety (WO 02/088172). Exemplary auristatin embodiments include theN-terminus linked monomethylauristatin drug moieties DE and DF,disclosed in “Monomethylvaline Compounds Capable of Conjugation toLigands,” U.S. Pat. No. 7,498,298. As used herein, the abbreviation“MMAE” refers to monomethyl auristatin E. As used herein theabbreviation “MMAF” refers todovaline-valine-dolaisoleuine-dolaproine-phenylalanine.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lubke, “The Peptides,”volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry.

Maytansinoids may be used as an active agent coupled to the antibody orfragment thereof according to the invention. Maytansinoids are mitototicinhibitors which act by inhibiting tubulin polymerization. Maytansinewas first isolated from the east African shrub Maytenus serrata (U.S.Pat. No. 3,896,111). Subsequently, it was discovered that certainmicrobes also produce maytansinoids, such as maytansinol and C-3maytansinol esters (U.S. Pat. No. 4,151,042). Highly cytotoxicmaytansinoid drugs can be prepared from ansamitocin precursors producedby fermentation of microorganisms such as Actinosynnema.Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020. An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources.

Selected examples of the calicheamicin family of antibiotics may be usedas an active agent coupled to the antibody or fragment thereof accordingto the invention. The calicheamicin family of antibiotics is capable ofproducing double-stranded DNA breaks at sub-picomolar concentrations.For the preparation of conjugates of the calicheamicin family, see U.S.Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,5,770,710, 5,773,001, 5,877,296. Another anti-tumor drug that theantibody can be conjugated is QFA which is an antifolate. Bothcalicheamicin and QFA have intracellular sites of action and do notreadily cross the plasma membrane. Therefore, cellular uptake of theseagents through antibody mediated internalization greatly enhances theircytotoxic effects.

Other antitumor agents that can be conjugated to the antibodies includeBCNU, streptozoicin, vincristine and 5-fluorouracil, the family ofagents known collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom.

A pharmaceutically acceptable carrier in the sense of the presentinvention may be any non-toxic material that does not significantlyinterfere in a detrimental sense with the effectiveness of thebiological activity of the antibodies of the present invention.Evidently, the characteristics of the carrier will depend on the routeof administration. Such a composition may contain, in addition to theactive substance and carrier, diluents, fillers, salts, buffers,stabilizers, solubilizers, and other materials well known in the art.Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintra-muscular administration and formulation.

The medicament, otherwise known as a pharmaceutical composition,containing the active ingredient (antibody or antibody fragment) may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example corn starch, or alginic acid; bindingagents, for example starch, gelatin or acacia, and lubricating agents,for example magnesium stearate, stearic acid or talc. The tablets may beuncoated or they may be coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. They may also be coated. The present invention also refersto a pharmaceutical composition for topical application, oral ingestion,inhalation, or cutaneous, subcutaneous, or intravenous injection. Askilled person is aware of the carriers and additives required forparticular application forms.

When a therapeutically effective amount of the active substance(antibody or antibody fragment) of the invention is administered byintravenous, cutaneous or subcutaneous injection, the active substancemay be in the form of a pyrogen-free, parenterally acceptable aqueoussolution.

The invention also relates to administration of a therapeuticallyrelevant amount of antibody as described herein in the treatment of asubject who has the medical disorders as disclosed herein. As usedherein, the term “therapeutically effective amount” means the totalamount of each active component of the pharmaceutical composition ormethod that is sufficient to show a meaningful patient benefit. Theamount of active substance in the pharmaceutical composition of thepresent invention will depend upon the nature and severity of thecondition being treated, and on the nature of prior treatments which thepatient has undergone. Larger doses may be administered until theoptimal therapeutic effect is obtained for the patient, and at thatpoint the dosage is not increased further.

The preparation of such parenterally acceptable solutions, having dueregard to pH, isotonicity, stability, and the like, is within the skillin the art. A preferred pharmaceutical composition for intravenous,cutaneous, or subcutaneous injection should contain, in addition to theactive substance, an isotonic vehicle such as Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, Lactated Ringer's Injection, or other vehicle as known in theart. The pharmaceutical composition of the present invention may alsocontain stabilizers, preservatives, buffers, antioxidants, or otheradditives known to those of skill in the art.

The dose of the antibody administered evidently depends on numerousfactors well-known in the art such as, e.g., the chemical nature andpharmaceutical formulation of the antibody, and of body weight, bodysurface, age and sex of the patient, as well as the time and route ofadministration. For an adult, the dose may exemplarily be between 0.001μg and 1 g per day, preferably between 0.1 μg and 100 mg per day, morepreferably between 1 μg and 100 mg per day, even more preferably between5 μg and 10 mg per day. In a continuous infusion, the dose mayexemplarily be between 0.01 μg and 100 mg, preferably between 1 μg and10 mg per kilogram body mass per minute.

In another aspect of the present invention there is provided an antibodyaccording to the invention as herein described for use in the treatmentof a B-cell mediated or plasma cell mediated disease or antibodymediated disease or disorder selected from Multiple Myeloma (MM),chronic lymphocytic leukemia (CLL), Non-secretory multiple myeloma,Smoldering multiple myeloma, Monoclonal gammopathy of undeterminedsignificance (MGUS), Solitary plasmacytoma (Bone, Extramedullar),Lymphoplasmacytic lymphoma (LPL), Waldenstrom's Macroglobulinemia,Plasma cell leukemia, Primary Amyloidosis (AL), Heavy chain disease,Systemic lupus erythematosus (SLE), POEMS syndrome/osteoscleroticmyeloma, Type I and II cryoglobulinemia, Light chain deposition disease,Goodpasture's syndrome, Idiopathic thrombocytopenic purpura (ITP), Acuteglomerulonephritis, Pemphigus and Pemphigoid disorders, andEpidermolysis bullosa acquisita; or any Non-Hodgkin's Lymphoma B-cellleukemia or Hodgkin's lymphoma (HL) with BCMA expression or any diseasesin which patients develop neutralising antibodies to recombinant proteinreplacement therapy wherein said method comprises the step ofadministering to said patient a therapeutically effective amount of theantibody as described herein.

B-cell disorders can be divided into defects of B-celldevelopment/immunoglobulin production (immunodeficiencies) andexcessive/uncontrolled proliferation (lymphomas, leukemias). As usedherein, B-cell disorder refers to both types of diseases, and methodsare provided for treating B-cell disorders with an antibody.

In one aspect of the present invention the disease is Multiple Myeloma.

Use of the antibody as described herein in the manufacture of amedicament for the treatment of diseases and disorders as describedherein is also provided.

For example in one aspect of the invention there is provided the use ofthe antibody as described herein for use in the treatment or prophylaxisof diseases and disorders responsive to modulation (such as inhibitingor blocking) of the interaction between BCMA and the ligands BAFF andAPRIL.

In one embodiment of the invention the isolated antibody or antibodyfragment is intended for use in the treatment of B lymphocyte cancers,such as Hodgkin's lymphoma.

In one embodiment of the invention the isolated antibody or antibodyfragment is intended for use in the treatment of an autoimmune disease,such as a medical disorder associated with inflammation, preferablyautoimmune disease with an inflammatory component, whereby theautoimmune disease is selected from Takayasu Arteritis, Giant-cellarteritis, familial Mediterranean fever, Kawasaki disease, Polyarteritisnodosa, cutanous Polyarteritis nodosa, Hepatitis-associated arteritis,Behcet's syndrome, Wegener's granulomatosis, ANCA-vasculitidies,Churg-Strauss syndrome, microscopic polyangiitis, Vasculitis ofconnective tissue diseases, Hennoch-Schõnlein purpura, Cryoglobulinemicvasculitis, Cutaneous leukocytoclastic angiitis, Tropical aortitis,Sarcoidosis, Cogan's syndrome, Wiskott-Aldrich Syndrome, Lepromatousarteritis, Primary angiitis of the CNS, Thromboangiitis obliterans,Paraneoplastic ateritis, Urticaria, Dego's disease, Myelodysplasticsyndrome, Eythema elevatum diutinum, Hyperimmunoglobulin D, AllergicRhinitis, Asthma bronchiale, chronic obstructive pulmonary disease,periodontitis, Rheumatoid Arthritis, atherosclerosis, Amyloidosis,Morbus Chron, Colitis ulcerosa, Autoimmune Myositis, Diabetes mellitus,Multiple sclerosis, Guillain-Barre Syndrome, histiocytosis,Osteoarthritis, atopic dermatitis, periodontitis, chronicrhinosinusitis, Psoriasis, psoriatic arthritis, Microscopic colitis,Pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still'sdisease, erythema nodosum, otitis, cryoglobulinemia, Sjogren's syndrome,Lupus erythematosus, aplastic anemia, Osteomyelofibrosis, chronicinflammatory demyelinating polyneuropathy, Kimura's disease, systemicsclerosis, chronic periaortitis, chronic prostatitis, idiopathicpulmonary fibrosis, chronic granulomatous disease, Idiopathic achalasia,bleomycin-induced lung inflammation, cytarabine-induced lunginflammation, Autoimmunthrombocytopenia, Autoimmunneutropenia,Autoimmunhemolytic anemia, Autoimmunlymphocytopenia, Chagas' disease,chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto'sThyroiditis, atropic thyroiditis, Graves disase, Autoimmunepolyglandular syndrome, Autoimmune Addison Syndrome, Pemphigus vulgaris,Pemphigus foliaceus, Dermatitis herpetiformis, Autoimmune alopecia,Vitiligo, Antiphospholipid syndrome, Myasthenia gravis, Stiff-mansyndrome, Goodpasture's syndrome, Sympathetic ophthalmia, Folliculitis,Sharp syndrome and/or Evans syndrome, in particular hay fever,periodontitis, atherosclerosis, rheumatoid arthritis, preferablyrheumatoid arthritis or multiple sclerosis.

SEQUENCES

Preferred Antibody Sequences of the Invention:

SEQ ID No. Sequence Description SEQ ID No. 1QVQLQQSGGGLVQPGGSLKLSCAASGIDFSRYWMSWVR HC (VH) mouseRAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCASLYYDYGDAMDYWGQGT SVTVSS SEQ ID No. 2EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYWMSWVR HC partiallyQAPGKGLEWVGEINPDSSTINYAPSLKGRFTISRDNAK humanizedNTLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWGQGT LVTVSS SEQ ID No. 3EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVR hHC01QAPGKGLVWVGEINPDSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWGQGT LVTVSS SEQ ID No. 4EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWX ₁SWV hHC02 RQAPGKGLVWVGEINPX ₂ X₃STINYAPSLKDKFTISRD NAKNTLYLQMNSLRAEDTAVYYCASLYX ₄DYGDAX ₅DY WGQGTLVTVSSWherein X₁: I, F, L, V, Y. C, G, A, S, T,  preferably I or F;X₂X₃: SS, NS, TS, GS, KS, RS, SD, SN, DE, preferably SS;X₄: Y, L, A, V, F, I, W, preferably Y; and/orX₅: Y, L, F, I, V, A, C, preferably Y SEQ ID No. 5EVQLVESGGGLVQPGGSLRLSGAASGFTFSRYX ₁MX ₂ W hHC03 VRQAPGKGLVX ₃VGX ₄INPDSSTINYAPSLKDKFTISR DNAKNTLYLQMNSLRAEDTAVYYCASX ₅ X ₆ X ₇DYGDX ₈MDYWGQGTLVTVSS Wherein X₁: W, F, Y, preferred W;X₂: S, T, N, Q, D, E, preferred S; X₃: W, F, Y, preferred W;X₄: E, Q, preferred E; X₅: L, I, V, G, A, preferred L;X₆: Y, X, preferred Y; X₇: Y, F, L, I, V, M, preferred Y; and/orX₈: A, G, V, preferred A SEQ ID No. 6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWhHC04 ISWVRQAPGKGLVWVGEINPNSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASL YYDYGDAYDYWGQGTLVTVSS SEQ ID No. 7EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYW hHC05FSWVRQAPGKGLVWVGEINPNSSTINYAPSLKD KFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGTLVTVSS SEQ ID No. 8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWhHC06 ISWVRQAPGKGLVWVGEINPSSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASL YYDYGDAYDYWGQGTLVTVSS SEQ ID No. 9EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYW hHC07FSWVRQAPGKGLVWVGEINPSSSTINYAPSLKD KFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGTLVTVSS SEQ ID No. 43 DIVMTQSQRFMTTSVGDRVSVTCKASQSVDSNVLC (VL) mouse AWYQQKPRQSPKALIFSASLRFSGVPARFTGSGSGTDFTLTISNLQSEDLAEYFCQQYNNYPLTFG AGTKLELKR SEQ ID No. 10DIVMTQSPATLSVSVGDEVTLTCKASQSVDSNVAWYQQ LC partiallyKPGQAPKLLIYSDDLRFSGVPARFSGSGSGTDFTLTIS humanizedSLQSEDFAVYYCQQYNNYPLTFGAGTKLELKR SEQ ID No. 11EIVMTQSPATLSVSPGERATLSCKASQSVDSNVAWYQQ hLC01 KPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTIS SLQSEDFAVYYCQQYNNYPLTFGAGTKLELKRSEQ ID No. 12 EIVMTQSPATLSVSPGERATLSCKASQSVX ₁ X ₂NVAWY hLC02QQKPGQAPRALIYSASLRFSGIPARESGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELKR Wherein:X₁X₂: ES, SS, TS, QS, HS, DH, preferably ES. SEQ ID No. 13EIVMTQSPATLSVSPGERATLSCKASQSVDX ₁ X ₂VX ₃ W hLC03 X ₄QQKPGQAPRALIX ₅ X₆AX ₇ X ₈RX ₉SGIPARFSG5X ₁₀ X ₁₁ GTEFTLTISSLQSEDFAVYYC X ₁₂QX ₁₃NNX ₁₄PX₁₅TFG AGTKLELKR Wherein: X₁: S, H, T, N, D, Q; X₂: N, E, Q;X₃: A, G, V, S, T, L, I; X₄: Y, F, L, I, V, A, G; X₅: Y, F, L; X₆: S, T;X₇: S, T, D, N, H, E, Q; X₈: L, V, I, M; X₉: F, L, I, V, Y, M;X₁₀: G, X; X₁₁: S, X; X₁₂: Q, V, L, I, M; X₁₃: Y, F, L, I, Q;X₁₄: Y, F, R, Q, K; and/or X₁₅: L, I, V, F SEQ ID No. 14EIVMTQSPATLSVSPGERATLSCKASQSVESNVAWYQQ hLC04KPGQAPRALIYSASLRFSGIPARESGSGSGTEFTLTIS SLQSEDFAVYYCQQYNNYPLIFGAGTKLELKRSEQ ID No. 15 RYWX ₁S H-CDR1 PTM Wherein:X₁: I, F, L, V, Y. C, G, A, S, T,  preferably I or F SEQ ID No. 16 EINPX₂ X ₃STINYAPSLKDK H-CDR2 PTM Wherein:X₂X₃: SS, NS, TS, GS, KS, RS, SD, SN, DE, preferably SS SEQ ID No. 17SLYX ₄DYGDAX ₅DYW H-CDR3 PTM Wherein:X₄: Y, L, A, V, F, I, W, preferably Y; and/orX₅: Y, L, F, I, V, A, C, preferably Y SEQ ID No. 18 RYWIS H-CDR1 PTM aSEQ ID No. 19 RYWFS H-CDR1 PTM b SEQ ID No. 20 EINPNSSTINYAPSLKDKH-CDR2 PTM a SEQ ID No. 21 EINPSSSTINYAPSLKDK H-CDR2 PTM b SEQ ID No. 22SLYYDYGDAYDYW H-CDR3 PTM a SEQ ID No. 23 KASQSVX ₁ X ₂NVA L-CDR1 PTMWherein: X₁X₂: ES, SS, TS, QS, HS, DH, preferably ES SEQ ID No. 24SASLRFS L-CDR2 PTM SEQ ID No. 25 QQYNNYPLTFG L-CDR3 PTM SEQ ID No. 26KASQSVDSNVA L-CDR1 PTM a SEQ ID No. 27EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVR Full lengthQAPGKGLVWVGEINPDSSTINYAPSLKDKFTISRDNAK humanized HCNTLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGOPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID No. 28 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWFSWVR Full lengthQAPGKGLVWVGEINPSSSTINYAPSLKDKFTISRDNAK humanized HC withNTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGT PTM mutations 1LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY (FSY)FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGOPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID No. 29 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWISWVR Full lengthQAPGKGLVWVGEINPSSSTINYAPSLKDKFTISRDNAK humanized HC withNTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGT PTM mutations 2LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY (ISY)FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGOPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID No. 30 EIVMTQSPATLSVSPGERATLSCKASQSVDSNVAWYQQ Full lengthKPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTIS humanized LCSLQSEDFAVYYCQQYNNYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGECSEQ ID No. 31 EIVMTQSPATLSVSPGERATLSCKASQSVESNVAWYQQ Full lengthKPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTIS humanized LC withSLQSEDFAVYYCQQYNNYPLTFGAGTKLELKRTVAAPS PTM mutationsVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

Preferred Nucleotide Sequences

SEQ ID No. 32 GAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTA Full lengthGTAGCAACTGCAACCGGTGTCCACAGTGAAGTGCAGCTGG humanized HCTCGAATCTGGAGGAGGCCTGGTTCAGCCTGGTGGCAGCCTTAGGCTCTCTTGTGCAGCCTCTGGCTTTACCTTCTCACGGTATTGGATGAGCTGGGTGAGACAGGCTCCAGGGAAAGGTCTGGTGTGGGTAGGGGAGATAAACCCCGATAGCAGCACGATCAACTATGCTCCGTCACTGAAAGACAAGTTCACCATTTCCCGCGATAATGCCAAGAACACTCTCTACTTGCAGATGAATTCCCTTCGAGCCGAGGATACAGCGGTGTACTACTGCGCCAGTCTGTACtacgactATGGGGACGCAATGGACTATTGGGGACAAGGCACACTGGTGACTGTTAGCTCCGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGAGTGCGACGGCCGGGCGGCGG CGGCGGATCC SEQ ID No. 33GAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTA Full lengthGTAGCAACTGCAACCGGTGTCCACAGTGAAGTGCAGCTGG humanized HC withTCGAATCTGGAGGAGGCCTGGTTCAGCCTGGTGGCAGCCT PTM mutations 1TAGGCTCTCTTGTGCAGCCTCTGGCTTTACCTTCTCACGGTATTGGTTCAGCTGGGTGAGACAGGCTCCAGGGAAAGGTCTGGTGTGGGTAGGGGAGATAAACCCCAGCAGCAGCACGATCAACTATGCTCCGTCACTGAAAGACAAGTTCACCATTTCCCGCGATAATGCCAAGAACACTCTCTACTTGCAGATGAATTCCCTTCGAGCCGAGGATACAGCGGTGTACTACTGCGCCAGTCTGTACTACGACTATGGGGACGCATACGACTATTGGGGACAAGGCACACTGGTGACTGTTAGCTCCGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGAGTGCGACGGCCGGGCGGCGG CGGCGGATCC SEQ ID No. 34GAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTA Full lengthGTAGCAACTGCAACCGGTGTCCACAGTGAAGTGCAGCTGG humanized HC withTCGAATCTGGAGGAGGCCTGGTTCAGCCTGGTGGCAGCCT PTM mutations 2TAGGCTCTCTTGTGCAGCCTCTGGCTTTACCTTCTCACGGTATTGGaTCAGCTGGGTGAGACAGGCTCCAGGGAAAGGTCTGGTGTGGGTAGGGGAGATAAACCCCAGCAGCAGCACGATCAACTATGCTCCGTCACTGAAAGACAAGTTCACCATTTCCCGCGATAATGCCAAGAACACTCTCTACTTGCAGATGAATTCCCTTCGAGCCGAGGATACAGCGGTGTACTACTGCGCCAGTCTGTACTACGACTATGGGGACGCATACGACTATTGGGGACAAGGCACACTGGTGACTGTTAGCTCCGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGAGTGCGACGGCCGGGCGGCGG CGGCGGATCC SEQ ID No. 35GAATTCCACCATGGGATGGtcATGTATCATCCTTTTTCTA Full lengthGTAGCAACTGCAACCGGTGTACACTCCGAGATCGTGATGA humanized LCCCCAGTCTCCTGCTACCCTGAGCGTTTCTCCCGGTGAAAGGGCCACACTCAGCTGCAAAGCCTCTCAAAGCGTGGACAGCAATGTCGCCTGGTATCAGCAGAAACCTGGCCAAGCTCCGAGAGCACTGATCTATTCCGCGTCATTGCGCTTTTCCGGCATACCAGCACGGTTTAGTGGCTCAGGGAGTGGGACTGAGTTCACTCTGACGATTAGCTCCCTTCAGTCAGAGGATTTCGCCGTGTACTACTGTCAGCAGTACAACAACTATCCCCTCACATTCGGAGCTGGAACCAAGCTGGAACTGAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGGGATCC SEQ ID No. 36GAATTCCACCATGGGATGGtcATGTATCATCCTTTTTCTA Full lengthGTAGCAACTGCAACCGGTGTACACTCCGAGATCGTGATGA humanized LC withCCCAGTCTCCTGCTACCCTGAGCGTTTCTCCCGGTGAAAG PTM mutationsGGCCACACTCAGCTGCAAAGCCTCTCAAAGCGTGGAGAGCAATGTCGCCTGGTATCAGCAGAAACCTGGCCAAGCTCCGAGAGCACTGATCTATTCCGCGTCATTGCGCTTTTCCGGCATACCAGCACGGTTTAGTGGCTCAGGGAGTGGGACTGAGTTCACTCTGACGATTAGCTCCCTTCAGTCAGAGGATTTCGCCGTGTACTACTGTCAGCAGTACAACAACTATCCCCTCACATTCGGAGCTGGAACCAAGCTGGAACTGAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGGGATCC

Preferred Sequences of the Invention Pertaining to CD269 (BMCA):

SEQ ID No. Sequence Description SEQ ID No. 37MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK GST-BCMA-HisWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNALEH HHHHH SEQ ID No. 38MAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNA BCMA extracellularSVTNSVKGTNALE domain SEQ ID No. 39MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRY BCMA N-terminusCNASVTNSVKGTNALE sequence SEQ ID No. 40 YFDSLLHACIPCQLRCSSNTBCMA antibody epitope - amino acids 13 to 32 of BCMA

Preferred Generalized Amino Acid Sequences Comprising the HumanizedSequence Modifications:

SEQ ID No. Sequence Description SEQ ID No. 41X₁VQLX₂X₃SGGGLVQPGGSLX₄LSCAASGX₅X₆FX₇X₈YWZ₁ General sequence forSWVRX₉APGKGLEWX₁₀GEINPZ₂SSTINYAPSLKX₁₁X₁₂F humanized HCX₁₃ISRDNAKNTLYLQMX₁₄X₁₅X₁₆RX₁₇EDTAX₁₈YYCASLY antibodies comprisingYDYGDAZ₃DYWGQGTX₁₉VTVSS the PTM deletionwherein X1: Q, E; X2: Q, V; X3: Q, E;  modificationsX4: K, R; X5: I,F; X6: D, T; X7: S, D; X8: R, D; X9: R, Q; X10: I, V; X11: D, G; X12: K, R; X13: I, T; X14: S, N; X15: K, S; X16: V, L; X17: S, A; X18: L, V; X19: S, L; and wherein at leastone of Z₁: I or F; Z₂: S and/or Z₃: Y. SEQ ID No. 42X₁IVMTQSX₂X₃X₄X₅X₆X₇SVGBX₈VX₉X₁₀TCKASQSVESNV General sequence forAWYQQKPX₁₁QX₁₂PKX₁₃LIX₁₄SX₁₅X₁₆LRFSGVPARFX₁₇G humanized LCSGSGTDFTLTISX₁₈LQSEDX₁₉AX₂₀YX₂₁CQQYNNYPLTF antibodies comprisingGAGTKLELKR the PTM deletion wherein X1: D, E; X2: Q, P; X3: R, A; modifications X4: F, T; X5: M, L; X6: T, S; X7: T, V; X8: R, E; X9: S, T; X10: V,L; X11: R, G; X12: S, A; X13: A, L; X14: F, Y; X15: A,   D; X16: S, D; X17: T, S; X18: N, S;  X19: L, F; X20: E, V; X21: F, Y

Examples

The invention is demonstrated by way of the examples disclosed herein.The examples provided herein represent only particular embodiments ofthe invention and are not intended to limit the scope of the invention.The examples are to be considered as providing a further description ofpossible and potentially preferred embodiments that enhance thetechnical support of one or more non-limiting embodiments.

Although the examples with respect to crystallization of theantibody-epitope complex and the in vitro and in vivo anti-tumor effectwas carried out using the original chimeric antibody J22.9-xi, theinventors assert that these technical effects are maintained in thehumanized variants of the present invention, due to the maintenance ofbinding characteristics in the humanized variants compared to theoriginal chimeric antibody tested. The data provided from the chimericantibody is therefore provided as reference material and an indicationof the industrial applicability and usefulness of the claimed humanvariants. Preliminary biological data indicates comparable effectsbetween J22.9-xi and the humanized variants.

Binding and Blocking Characteristics of the J22.9-xi and BCMAInteraction

The novel chimeric antibody (J22.9-xi) binds to the extracellular domainof human CD269 (BCMA, TNFRSF17). This was initially ascertained by ELISAand flow cytometry on the human multiple myeloma cell line MM.1S (FIG. 1a,b ). The affinity of J22.9-xi to BCMA was determined using surfaceplasmon resonance (SPR). The mean Kd is 54 pM as shown in FIG. 1 c.

BCMA is known to trigger signals important for the survival of multiplemyeloma and plasma cells in vivo through interaction with its ligandsBAFF and/or APRIL (Mackay F et al. (2003) Annu Rev Immunol 21:231-264).An in vitro blocking assay was therefore performed with theextracellular domain of human BCMA and recombinant BAFF. The binding ofJ22.9-xi to BCMA clearly blocks the interaction between the receptor andits ligand BAFF. Using the isotype control antibody instead of J22.9-xi,recombinant BAFF binding to BCMA is unaffected (FIG. 1 d ).

The Crystal Structure of the J22.9-xi-Fab-BCMA-Complex Reveals anExtensive Binding Interface with BCMA

Fab fragments prepared from J22.9-xi were crystallized in complex withthe purified 46 amino acid residue BCMA extracellular domain and thecomplex structure solved to 1.9 angstroms resolution. High qualityelectron density is observable for residues 6 to 41 of BCMA and shows anextensive interaction with J22.9-xi, primarily with the light chain ofthe antibody (FIG. 2B). This interface, which buries 740.4 squareangstroms and involves one third of the BCMA residues, covers 12 of 16residues of the identical epitope observed in the crystal structures ofBMCA complexes with APRIL and sTALL1 (also known as BAFF), including theconserved DxL motif (Gordon N C, et al. (2003), BAFF/BlysS receptor 3comprises a minimal TNF receptor-like module that encodes a highlyfocused ligand-binding site. Biochemistry 42(20): 5977-83, and Patel DR, et al. (2004), Engineering an APRIL-specific B Cell MaturationAntigen. JBC 279(16): 16727-35), providing clear rationalization of theblocking effect seen in the in vitro assays with BAFF (FIG. 2A). Theinteraction with J22.9-xi additionally comprises a direct side chaincontact with Ala20 and Pro23 in BCMA, residues not part of the bindingepitope covered by BAFF and APRIL, and several water-mediated hydrogenbonds. The overall conformation of BCMA in all three structures is verysimilar, with a C-alpha rmsd of 1.4 angstroms between the J22.9-xi andAPRIL complexes and 1.5 angstroms between the J22.9-xi and sTALL1complexes; the respective C-alpha rmsds for the J22.9-xi BCMA bindingepitope (residues 13-30) are 0.98 and 0.88 angstroms. Althoughrecognizing the same BCMA epitope having the DxL motif at its core, thebinding site of J22.9-xi is very different from those of sTALL1 andAPRIL, as is the collection of interactions comprising the interface.

As can be seen in FIG. 2B and Tables 1 and 2, 19 amino acids fromJ22.9-xi (6 from the heavy chain (Table 1), 13 from the light chain(Table 2)) form direct linkages to 12 residues from the extracellulardomain of CD269.

TABLE 1 Amino acid interaction list between heavy chain of J22.9-xi andBCMA. These interaction lists were generated using the software PDBsum(Laskowski R A (2009)). Heavy chain CD269 Trp33 > His19 Glu50 > His19Leu99 > Leu17 > Leu18 Tyr100 Leu18 Tyr101 > Ala20 > Ile22 > Pro23Ala106 > Leu18

TABLE 2 Amino acid interaction list between light chain of J22.9-xi andBCMA. These interaction lists were generated using the software PDBsum(Laskowski R A (2009)). Light chain CD269 Ser31 > Arg27 Thr32 Ala34 >Leu17 Tyr36 > Leu17 Phe49 > Leu18 Asp15 Ser50 > Tyr13 Asp15 Arg27Ser52 > Arg27 Ser67 > Thr32 Leu53 > Tyr13 Leu26 Arg27 Phe55 > Leu18Gln89 > Leu17 Tyr91 > Asp15 Ser16 Leu17 Tyr94 > His19 Leu96 > Leu17

TABLE 3 Interaction list of the residues involved in CD269:APRIL undCD269:BAFF binding (residues NOT directly contacted by J22.9 areunderlined). These interaction lists were generated using the softwarePDBsum (Laskowski R A (2009)). APRIL CD269 Asp121 Leu35 Asp123 Pro33Pro34 Leu35 Asp164 Asn31 Thr166 Arg27 Ser30 Phe167 Tyr13 Leu18 Ile22Leu26 Arg27 Asn31 Thr168 Leu18 Leu26 Met169 Leu17 Gly170 Leu17 His19Gln171 Leu17 Arg186 Leu17 Leu18 His19 Cys187 Leu17 Ile188 Asp15 Leu17Leu18 Asp196 Leu26 Arg197 Leu26 Tyr199 Leu18 Pro221 Leu17 His19 Arg222Asp15 Leu17 Arg27 Asn224 Thr32 Lys226 Asn31 His232 His19 BAFF CD269Tyr22 Ser16 Asp62 Asn31 Lys63 Ser30 Asn31 Thr64 Arg27 Ser30 Asn31 Tyr65Tyr13 Asp15 Leu18 Ile22 Ala66 Leu17 Met67 Leu17 Gly68 Leu17 Arg90 Leu17His19 Cys91 Leu17 Ile92 Leu17 Leu18 Glu97 Ser29 Ser30 Asn31 Leu99 Ile22Leu26 Asn101 Leu18 Pro123 Ser16 Leu17 Arg124 Tyr13 Asp15 Leu17 Arg27Glu125 Arg27 Thr25 Pro34 Leu35 Asn126 Thr32 Asp132 His19

TABLE 4 Residues of the CD269 target bound by direct contacts of J22.9,APRIL and/or BAFF. Residues of the CD269 target directly contacted onlyby J22.9 are underlined (20, 23). Residues of the CD269 target NOTdirectly contacted by J22.9 are in bold type (30, 31, 33, 34, 35 forAPRIL; 25, 29, 30, 31, 34, 35 for BAFF). J22.9: 13, 15, 16, 17, 18, 19,20, 22, 23, 26, 27, 32 APRIL: 13, 15, 17, 18, 19, 22, 26, 27, 30, 31,32, 33, 34, 35 BAFF: 13, 15, 16, 17, 18, 19, 22, 25, 26, 27, 29, 30, 31,32, 34, 35

TABLE 5 J22.9 Water interactions (J22.9-xi:H2O:CD269). The data in table5 was generated using the software LigPlot (Wallace and Laskowski,European Bioinformatics Institute). (sc = side chain H-bond; mc = mainchain H-bond) Light Chain H₂O# CD269 Ser31 (sc) 285 Thr32 (sc, mc) 285,286 Arg27 (sc) 285, 286 Ser30 (sc) Ser31 (mc) 283, 284 Arg27 (sc) Asn32(sc) 105 Asp15 (sc) 105, 284 Arg27 (sc) 56 Ser16 (sc) Tyr36 66, 93, 450Leu17 (mc) Ser50 (sc) 105 Asp15 (sc) Ser52 (sc) 286 Ser30 (sc) 286 Arg27(sc) 286, 285 Thr32 (sc, mc) Gly66 (mc) 287 Thr32 (sc) 285, 286 Arg27(sc) 285, 286 Ser30 (sc) Gln89 (sc) 66, 93, 450 Leu17 (mc) Tyr91 (mc)282 Ser16 (sc) 282, 281 Ser16 (mc) Tyr94 (sc) 281 Ser16 (mc) 281, 282Ser16 (sc) Heavy Chain H₂O# CD269 Trp33 (mc) 42, 280 Leu17 (mc) 183,279, 26 Leu18 (mc) Ser35 (sc) 42, 66, Leu17 (mc) 93, 280, 450 Trp47 (sc)93, 450 Leu17 (mc) Glu50 (sc) 281 Ser16 (mc) 281, 282 Ser16 (sc) 450Leu17 (mc) 450, 280 Leu17 (mc) Leu99 (mc) 280 Leu17 (mc) Tyr101 (mc) 26Leu18 (mc)

Strong Cytotoxic Efficacy of J22.9-xi is Strongly Decreased afterDeglycosylation

A luciferase-based cytotoxicity assay was established using theluciferase transduced MM.1S-Luc cell line. In this assay,bioluminescence is only detected from living cells since luciferasereleased by dead cells is unable to function due to the lack of ATP inthe medium. PBMCs from healthy donors were isolated and mixed withMM.1S-Luc cells in a ratio of 20 to 1. After 4 hours the bioluminescencewas measured.

With a selection of 4 unstimulated donor PBMC preparations, the in vitrocytotoxicity of J22.9-xi was determined. The cytotoxic potential variesslightly between PBMCs from different donors. Within 4 hours ofincubation, cell lysis reached 18 to 35% at a concentration of 125 ng/mlJ22.9-xi. Increasing the J22.9-xi concentration to 1 ug/ml increasedcell lysis up to 56% (FIG. 3 a ).

After deglycosylation of J22.9-xi (J22.9-xi-N-glycan) with PNGase F, thecytotoxic activity dropped to below 8%, whereas the binding ofJ22.9-xi-N-glycan to BCMA-positive MM.1S cells remained unaltered (FIG.3 a,b ).

J22.9-xi Reduces Tumor Burden in Xenografted Mice and Prolongs Survival

We used NOD scid common gamma chain knock out (NSG) mice lackingfunctional B, T and NK cell populations. These mice, injected with 1*10⁷MM.1S-Luc cells intravenously, develop hind limb paralysis within 6weeks (FIG. 4 d -1). The day on which the first symptom appears, definesthe day of killing.

After injection of 1×10⁷ MM.1S-Luc in the tail vein, the mice weredivided randomly into 3 groups. The first group (n=2) received notreatment until the end of the experiment, whereas the second (n=5) andthe third (n=6) group received twice weekly injections of 200 μg of anisotype control or the J22.9-xi antibody, respectively. The antibodieswere administered for a period of 6 weeks intraperitoneally (i.p.)starting with the day of tumor cell injection. Tumor growth wasmonitored once a week using the IVIS Spectrum. Bioluminescence wasmeasured 3 minutes after i.p. injection of luciferin.

A similar course of tumor development was seen in both the untreatedgroup and the group receiving the control antibody, whereas the grouptreated with J22.9-xi showed significantly less tumor burden, alreadybeginning at the first measurement point at day six (FIG. 4 a ). Inaddition, this group showed a smaller overall tumor load during thewhole monitoring period (FIG. 4 b ). Isotype control treated animals hada median survival of 46 days after cell injection. Mice receivingJ22.9-xi lived an average of 26 days longer. This corresponds to anextended survival of 55% compared with mice receiving the controlantibody (FIG. 4 c ). Massive infiltrations of tumor cells into thespine and inguinal lymph nodes were seen in non-treated mice and in micereceiving the isotype control antibody by day 28 after cell injection(FIG. 4 d -2).

Administration of 200 μg of an antibody to a mouse corresponds toapproximately 10 mg/kg bodyweight. To test the efficacy of J22.9-xi atlower doses we divided MM.1S-Luc-xenografted mice into four groups. Thefirst group (n=7) received 200 μg of the control antibody twice weekly,and groups 2, 3 (each n=3) and 4 (n=9) were injected with 2 μg, 20 μg or200 μg twice a week, respectively. Injection and monitoring wereperformed as described above.

Although tumors developed as expected in the control group mice,dramatically restricted tumor growth was observed in the groupsreceiving 20 μg or 200 μg of J22.9-xi (FIG. 4 e,f ). An overview of theexperimental timeline is provided in FIG. 4 g.

Growth of Established Tumors Arrests for 5 Weeks During J22.9-xiTreatment

Therapeutic administration was mimicked by delaying the start ofantibody treatment to 5 days after tumor cell injection. The xenograftedmice were divided into 2 groups (n=6). The animals received 200 μg perinjection of either the isotype control or J22.9-xi antibody twice aweek. The first measurement was done at day 8 post cell injection. Whilethere is no tumor-derived bioluminescence measurable to day 35 in thegroup receiving J22.9-xi (n=5), a steady increase in tumor load was seenin animals receiving the isotype control antibody (n=6) (FIG. 5 a,b ).Mice from the isotype control group survived an average of 56 days afterthe cell injection, whereas all mice receiving J22.9-xi are still aliveat day 77 (FIG. 5 c ). An overview of the experimental timeline isprovided in FIG. 5 d.

Intensive Early Phase Treatment with J22.9-xi Prevents Tumor Growth for7 Weeks

In order to further assess the effect of treatment timing on tumorgrowth, different antibodies were administered for five consecutive daysstarting from the day of tumor cell injection. Subsequent to i.v. cellinjection, the animals were divided randomly into 5 groups. Group 1(n=5) was treated with 200 μg of the isotype control antibody perinjection (i.p.), whereas group 2 (n=6) received 200 μg/injection of theJ22.9-xi-N-glycan antibody. The mice from groups 3 (n=4), group 4 (n=5)and group 5 (n=5) obtained 200 μg, 20 μg and 2 μg per injection of theJ22.9-xi antibody, respectively. Bioluminescence measurements began atday 9 post cell injection. Up to day 44, no tumor-derivedbioluminescence was seen in any of the groups receiving the intactJ22.9-xi antibody. Although the tumor growth in the animals treated withJ22.9-xi-N-glycan is decelerated, the overall tumor load is notsignificantly different from those animals receiving the isotype controlantibody (FIG. 6 a,b ). Although the overall tumor load of animalstreated with J22.9-xi-N-glycan (deglycosylated) was not significantlydifferent (FIG. 6 b ), the lifespan of these mice was substantiallyincreased compared to the isoAb-treated group (FIG. 6 c ). SinceJ22.9-xi-N-glycan was shown to be unable to induce ADCC or CDC, thisresult indicates that alone the binding of J22.9-xi to BCMA hinderstumor growth. It may be reasonably considered that this is due toblocking of the interaction between the receptor and its native ligands(APRIL and BAFF). An overview of the experimental timeline is providedin FIG. 6 d.

Humanisation of J22.9-xi

The J22.9-xi antibody was humanized based on sequence alignment and thedata obtained from the crystal structure. The sequences of the variableregions were aligned to their respective human homologs using IgBLAST(NCBI) or Clustal (EBI). Each proposed mutation was evaluated by visualinspection of the structure before alteration.

Binding of Humanized Variants to BCMA Target

Binding of the mutants to BCMA was tested using flow cytometry, ELISAand SPR. The affinity of the humanized antibodies was measured usingsurface plasmon resonance (ProteOn™ XPR36; Bio-Rad). The binding datashow surprising results with respect to the specificity and affinity ofthe humanized antibody variants to the same epitope as tested forJ22.9-xi binding. As shown in the table below, it was entirelysurprising that the humanized antibodies as described herein exhibitedcomparable binding characteristics as the original chimeric antibody.The SPR data reveals that the affinities of the humanized variants aresimilar to those of the chimera and are sufficient to assume theirclinical relevance in light of the data provided herein for the originalchimeric antibody. A skilled person would not have expected that throughthe modification of the CDRs during humanization of the chimera that thebinding characteristics would be maintained to such an extent.

ELISA was carried out as described herein using BCMA-coated microtiterplates (1 microg/ml). As observed in FIG. 10 , binding was comparablefor all humanized variants in comparison to J22.9-xi using both humanand cynomlgous BCMA.

Flow cytometry was also carried out using the humanized variantsdescribed herein and equivalent binding to both human and cynomlgousBCMA for all humanized variants tested was shown (refer FIG. 11 ).

SPR analysis was also conducted and affinities measured for humanizedantibody variants. As can be observed in the table below (table 6), theaffinities of the humanized variants (J22.9-H corresponds to humanizedsequence SEQ ID No. 27; J22.9-FSY corresponds to humanized and PTMmodified SEQ ID No. 28; J22.9-ISY corresponds to humanized and PTMmodified SEQ ID No. 29).

TABLE 6 SPR Data Affinity (SPR) Affinity (SPR) ELISA ELISA Flow Melting(human) (cynomolgous) Name (human) (cynomolgous) cytometry temperatures(n = 3) (n = 2) J22.9-xi +++ +++ +++ 86/94° C. 2.8 ± 0.7 × 2.7 × 10⁻⁹M10⁻¹⁰M J22.9-H ++ + nd 86/94° C. 1.5 ± 0.3 × 2.0 × 10⁻⁷M 10⁻⁸M J22.9-+++ +++ +++ 87/94° C. 2.2 ± 0.3 × 2.0 × 10⁻⁸M FSY 10⁻⁹M J22.9- +++ ++++++ 86/94° C. 2.0 ± 0.2 × 1.7 × 10⁻⁸M ISY 10⁻⁹M

Amino Acid 54 in CDR2 of the J22.9 Heavy Chain:

In order to remove a potential post-translational modification site inthe humanized J22.9, residue D54 of the heavy chain CDR2 was mutated toasparagine (N), inadvertently creating a new potential modification sitefor N-linked glycosylation. The mutated heavy chain containing N54migrated slower on SDS gels (FIG. 13 ), indicating a larger size andthat the CDR was glycosylated.

The corresponding IgG, J22.9-FNY, nevertheless bound BCMA in FACS andELISA, and was crystallized in complex with BCMA. Although notcompletely refined, the 2.7 Angstrom resolution structure shows clearelectron density extending from the N54 side chain —consistent with asugar modification of the residue. It is surprising that such a largeextension of the side chain would not disrupt binding to BCMA and itcould be expected from these observations that multiple and variousamino acid substitutions would be tolerated at this position,potentially also derivatizations other than sugars.

Methods

Cell Lines and Culture

The human multiple myeloma cell line MM.1S (Greenstein et al. (2003) ExpHematol 31:271-282) was obtained from Prof. B. Dörken (MDC, Berlin,Germany). For in vivo monitoring of tumor cell growth, Luciferase andGFP were cloned into the pFU vector of the lentiviral vector systemViraPower (Invitrogen). Via GFP-expression of transduced cells,monoclonal cell lines were isolated using fluorescence-activated singlecell sorting. Cell lines were cultured in RPMI-1640 medium withoutphenol red, containing 10% fetal calf serum, 100 units/ml of penicillin,and 100 μg/ml of streptomycin (all from PAA).

The HEK293-6E cells, purchased from the National Research Council ofCanada, were maintained in Freestyle F17 medium (Invitrogen)supplemented with 7.5 mM L-Glutamine (PAA), 0.1% Pluronic F-68(Invitrogen), and 25 μg/ml G418 (Invitrogen). Cells were grown inErlenmeyer flasks (Corning) at 110 rpm and 37° C. in a 5% CO2atmosphere.

Antibody Production and Purification

To obtain a BCMA-binding antibody, standard hybridoma technique wasused. 4 BL/6 wild type mice were immunized 6 times with incompleteFreund's adjuvant and 30 μg of the extracellular domain of human BCMAC-terminally fused to Glutathione S-transferase (GST). After cell fusionfollowed by a screening period the J22.9 hybridoma was shown to secretean anti-BCMA antibody.

Due to the instability of the hybridomas the variable regions of thelight and heavy chain of hybridoma J22.9 were amplified and clonedupstream of the human kappa or the IgG1 constant domain genes,respectively. The chimeric J22.9-xi antibody was produced by transientcotransfection of 293-6E cells with a 1:2 DNA plasmid mixture encodingthe light and heavy chains, respectively. In brief: 293-6E cells wereresuspended to 1.7×10⁶ cells/ml in serum free Freestyle F17 medium andtransfected using polyethyleneimine at a final concentration of 1 μg/mlculture. Two days after transfection, cells were fed with 100% of thetransfection volume Freestyle F17 medium containing 1% tryptone N1(Organo Technie). At day 7 cells were harvested by centrifugation andthe filtered (0.45 μm) culture medium was passed over a 3.5 ml Protein ASepharose column (Bio-Rad). The column was washed with 10 ml phosphatebuffered saline (PBS) and antibody eluted by addition of 20 mM sodiumacetate, 150 mM NaCl, pH 3.5. Fractions of 2 ml were collected directlyinto tubes containing 100 μl 1 M HEPES, pH 7.5 for neutralization. Thefinal yield of full length IgG was approximately 40 mg/I culture.

Since hybridoma J22.9 lost the capacity to produce/secrete the anti-BCMAantibody (FIG. 8 ), the variable regions of the heavy and light chainswere amplified using PCR and subsequently cloned at the 5′ end of thehuman constant IgG1 and K light chain genes, respectively. Throughco-transfection of 293-6E cells with these two plasmids, the chimericJ22.9-xi antibody was produced. The production of the antibody of theinvention was therefore inherently difficult and not achievable bystraightforward routine methods.

The isotype control antibody composed of the J22.9-xi heavy chain and arandom chimeric kappa light chain was produced in parallel with theJ22.9-xi antibody. This antibody was shown by ELISA and flow cytometryto be unable to bind to BCMA.

The N-linked oligosaccharide chains at Asn297 of the heavy chain ofJ22.9-xi were removed enzymatically using N-Glycosidase F (PNGase F)(NEB). 10 mg of J22.9-xi were incubated with 15,000 units PNGase F in500 μl PBS (pH 7.4) for 36 hours at 37° C. followed by buffer exchangeinto sterile PBS.

Determination of Binding and Blocking Capacities of J22.9-xi byEnzyme-Linked Immunosorbent Assays (ELISA)

Microtiter plates were coated with 10 μg/ml of the extracellular domainof human BCMA. Coated BCMA was detected with serial dilution of J22.9-xiand the isotype control ranging from 1 to 1000 ng. Binding of J22.9-xior isotype control antibody to the coated BCMA was detected withhorseradish peroxidase (HRP)-conjugated goat anti-human secondaryantibody (Jackson ImmunoResearch, 109-035-098, dilution 1:5,000).

Microtiter plates were coated with 1 μg/ml of the extracellular domainof human or cynomolgous BCMA (hBCMA or cyBCMA, respectively). CoatedBCMA was detected with serial dilution of J22.9-xi, J22.9-H, J22.9-ISYand J22.9-FSY ranging from 0.26 pM to 500 nM. Binding of antibodies tothe coated BCMA was detected with horseradish peroxidase(HRP)-conjugated goat anti-human secondary antibody (JacksonImmunoResearch, 109-035-098, dilution 1:5,000).

For the blocking experiment, 1 mg/ml of human recombinant BAFF fused toa His-tag (Biomol) was applied after the antibodies and washing anddetected using the mouse anti-His tag (AbD Serotec, AD1.1.10, dilution1:5,000, HRP-conjugated) antibody. All ELISAs were developed using BDOptEIA reagents A and B (BD Bioscience) and measured with a microplatespectrophotometer (BioTek) at 450 nm and 570 nm.

Flow Cytometry Analysis

For cell surface antigen detection experiments, self-made antibodies(J22.9-xi, J22.9-H, J22.9-ISY, J22.9-FSY and the isotype control) andcommercially available mouse anti-His tag (AbD Serotec, AD1.1.10,dilution 1:100, Alexa Fluor 488-conjugated) and goat anti-human IgG1(Jackson ImmunoResearch, 109-116-098, dilution 1:400, PE-conjugated)antibodies and human recombinant BAFF fused to a His-tag (Biomol) wereused. Experiments were performed on a FACSCalibur or a FACSCanto II flowcytometer (BD Bioscience). The data were analysed with Flowjo softwareversion 7.6 (TreeStar Inc.).

Generation of Fab and Fab:BCMA Complexes

(Fab)₂ fragments were generated from full length J22.9-xi IgG byincubation with pepsin. J22.9-xi was passed over a PD-10 buffer exchangecolumn into 50 mM sodium acetate, pH 3.5 and pepsin added at 30 μg permilligram J22.9-xi. Incubation at 37° C. for 2.5 hours was sufficient tocompletely digest the fragment crystallizable (Fc) region and pepsin wasinactivated by exchange over a PD-10 column into PBS (pH 7.2). Thereduction of the (Fab)₂ fragments to individual Fabs was accomplished inPBS by addition of 2-Mercaptoethylamine (50 mM) in the presence of 5 mMethylenediaminetetraacetic acid (EDTA). After incubation for 90 minutesat 37° C., the reduced cysteines were blocked by alkylation with 500 μMiodoacetamide for 30 minutes followed by buffer exchange into fresh PBS.The Fab fragments were combined with 1.5 molar equivalents of purifiedBCMA and the complexes isolated by size exclusion chromatography on aSuperdex 75 16/60 column. Fractions were analyzed on 4-12% SDSpolyacrylamide gels and fractions containing both Fab and BCMA werepooled and concentrated for crystallization trials.

Crystallization of Fab:BCMA Complexes

Concentrated complexes were supplemented with 0.5 molar equivalents ofpure BCMA to ensure saturation and were subjected to crystallizationscreening. Initial Fab:BCMA crystallization conditions were identifiedfrom commercial screens (Qiagen) in 96-well sitting drop format platesusing a Gryphon pipetting robot (200 nl drops) and optimized in 24 wellplates in hanging drops (2-3 ul). The complex was concentrated to 8mg/ml and crystallized in 21% PEG 3350, 0.1 M BisTris pH 6.5 and 5 mMCuCl₂ at 20° C. Crystals appeared after three days as clusters of thinplates and attained their final size (0.2-0.3 mm) within approximately 7days. Clusters were separated and individual plates were flash frozen inliquid nitrogen in mother liquor with 20% glycerol as cryoprotectant.Complete diffraction data was collected from a single crystal at theBESSY synchrotron of the Helmoltz Zentrum Berlin. The structure wassolved to a resolution of 1.9 angstroms by molecular replacement usingthe experimental phases from the structure of Efalizumab (3E09) as thesearch model. Data processing was performed with the ccp4 suite ofprograms, structure refinement was performed using Phenix (Adams P D, etal. (2010), Acta Cryst. D66: 213-221) and model building and assessmentusing Coot. (Emsley et al, Acta Crystallographica Section D—BiologicalCrystallography, 2010, 66:486-501) Images were made using PyMOL (ThePyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC).

In Vitro Cytotoxicity Assay

In this assay the cytotoxic effect of J22.9-xi was determined bymeasuring the luminescence of the remaining living cells in abioluminescence reader. In short: freshly obtained human filter buffycoats (FBC) were back-flushed by gravity with 160 ml elution buffer (PBS(pH 7.4) containing 5 mM Na₂-EDTA and 2.5 [w/v] sucrose). Mononuclearcells were isolated from the eluted cells by Ficoll gradientcentrifugation. Mononuclear cells from the interphases were taken andwashed twice in elution buffer. After erythrocyte lysis, PBMCs werewashed again, counted and adjusted by dilution in RPMI/10% FCS w/ophenol red to 1*10⁷ cells/ml. 5*10⁴ MM.1S-Luc cells in 50 μl RPMI wereplated in microtiter plates. Ten minutes prior to the addition of 100 μlPBMCs, the MM.1S-Luc cells were incubated with J22.9-xi or the isotypecontrol antibody serial dilutions in a sample volume of 200 μl. Afteraddition of target cells, antibodies and effector cells, microtiterplates were centrifuged (300×g) for 2 minutes at room temperature (RT)and stored at 37° C. with 5% CO2. Control wells were treated with 1%Triton X instead of antibody for complete lysis. After 4 hours ofincubation, 25 μl of PBS with luciferin (250 ng/ml) were applied to eachwell, and the bioluminescence of the living cells was measured in abioluminescence reader (Tecan). The specific cytotoxicity was calculatedaccording to the following formula:100−[value(J22.9-xi)−value(total lysis)]/[value(isotypecontrol)−value(total lysis)]*100.

In Vivo Studies

NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CSF2)2Ygy Tg(IL3)1YgyTg(KITLG)3YgyJGckRolyJ mice (NSG) from The Jackson Laboratory andCB17.Cg-Prkdcscid Lystbg/Crl mice from Charles River Deutschland(Sulzfeld, Germany) were used. Experiments were performed with micebetween 8-14 weeks old. All animal studies were performed according toinstitutional and state guidelines, under specific pathogen-freeconditions. In the experimental examples relating to treatment ofestablished tumours and tumour treatment in the early phase of diseasethe CB17.Cg-Prkdcscid Lystbg/Crl mice were used. The phenotype of thetwo mice strains mentioned herein is very similar. The animals have nofunctional B-, T- and NK-cells. A slightly slower tumour growth wasobserved in the CB17.Cg mice, indicating an even more promising effectof the therapeutic antibody of the present invention.

The xenograft model of multiple myeloma was induced by intravenousinjection of 1*10⁷ MM.1S-Luc cells in the tail vein at day zero. In thismodel, untreated animals develop hind limb paralysis within 6 weeks.Occurrence of this symptom indicates the end point of the experiment.

For the efficacy studies, the antibodies were administeredintraperitoneally (i.p.) twice a week or on 5 consecutive days startingat day zero. The J22.9-xi antibody was given in doses of 2 μg, 20 μg or200 μg per injection; for the isotype control antibody, 200 μg/injectionwas used. The bioluminescence of the MM.1S-Luc cells was measured afteri.p. injection of 150 μg luciferin using the IVIS Spectrum (Caliper LifeSciences). Measurements were done weekly. At each timepoint, 3 untreatedcontrol mice were also administered luciferin. Total flux values ofthese animals are either subtracted from each measurement or shown inthe graphs.

To treat established tumors, antibody therapy was begun 5 days afterinjection of the MM.1S-Luc cells. 200 μg of the J22.9-xi or isotypecontrol antibody was administered twice a week for a period of 6 weeks.

Humanization of J22.9-xi

The heavy and light chain variable region sequences (mouse) were alignedwith those from the corresponding heavy and light chain subtype humansequences to determine which residue alterations were required toproduce a fully humanized sequence variant. Using the crystal structureof the J22.9-xi:hBCMA complex, each modification was first assessed insilico to identify mutations that could potentially disrupt binding ofthe antibody to BCMA. Two complete J22.9 variable region genes for eachchain were synthesized, one with the original mouse sequence and onewith a completely humanized sequence (i.e. containing all of thenecessary humanizing mutations) with two added restriction enzyme sitesto divide the genes into three cassettes each. After flaggingpotentially problematic mutations, various combinations of the originalmouse and fully humanized gene cassettes were produced and theircorresponding IgGs were expressed, purified and subjected to FACSanalysis with BCMA positive cells to assess binding. Flagged problematicresidues were mutated individually using PCR to verify their effect onaffinity to BCMA and the final optimized constructs were subsequentlyquantitatively assessed for binding to both human and cynomolgus BCMAvia SPR.

SPR

SPR was performed on a ProteonXPR36 using phosphate buffered salinesupplemented with 0.005% Tween-20 (PBST). Whole IgG at a concentrationof 15 ug/ml was immobilized to a Proteon GLH sensor chip using standardamine chemistry according to the manufacturer's instructions. Forbinding experiments, human or cynomolgus BCMA in PBST was used as themobile phase. Binding affinities (K_(d)) were calculated fromassociation (k_(on)) and dissociation (k_(off)) constants determined inparallel at multiple concentrations of BCMA (ranging from 0.4 to 800 nMfor hBCMA and 2.7 nM to 1 μM for cynoBCMA) assuming a single-sitebinding model.

Additionally, further experimentation shows that the preferredembodiments of the invention provide surprising and unexpected effects,thereby solving the problem of the invention in a non-obvious fashion.

REFERENCES

-   Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I.    W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J.,    Grosse-Kunstleve, R. W., et al. (2010). PHENIX: a comprehensive    Python-based system for macromolecular structure solution. Acta    Crystallogr D Biol Crystallogr 66, 213-221.-   Al-Lazikani, B., Lesk, A. M., and Chothia, C. (1997). Standard    conformations for the canonical structures of immunoglobulins. J Mol    Biol 273, 927-948.-   Anthony, R. M., and Ravetch, J. V. (2010). A novel role for the IgG    Fc glycan: the anti-inflammatory activity of sialylated IgG Fcs. J    Clin Immunol 30 Suppl 1, S9-14.-   Chan et al. (2010) Nat Rev Immunol 10:301-316-   Chavez-Galan, L., Arenas-Del Angel, M. C., Zenteno, E., Chavez, R.,    and Lascurain, R. (2009). Cell death mechanisms induced by cytotoxic    lymphocytes. Cell Mol Immunol 6, 15-25.-   Chiu, A., Xu, W., He, B., Dillon, S. R., Gross, J. A., Sievers, E.,    Qiao, X., Santini, P., Hyjek, E., Lee, J. W., et al. (2007). Hodgkin    lymphoma cells express TACl and BCMA receptors and generate survival    and proliferation signals in response to BAFF and APRIL. Blood 109,    729-739.-   Gordon, N. C., Pan, B., Hymowitz, S. G., Yin, J., Kelley, R. F.,    Cochran, A. G., Yan, M., Dixit, V. M., Fairbrother, W. J., and    Starovasnik, M. A. (2003). BAFF/BLyS receptor 3 comprises a minimal    TNF receptor-like module that encodes a highly focused    ligand-binding site. Biochemistry 42, 5977-5983.-   Greenstein, S., Krett, N. L., Kurosawa, Y., Ma, C., Chauhan, D.,    Hideshima, T., Anderson, K. C., and Rosen, S. T. (2003).    Characterization of the MM.1 human multiple myeloma (MM) cell lines:    a model system to elucidate the characteristics, behavior, and    signaling of steroid-sensitive and -resistant MM cells. Exp Hematol    31, 271-282.-   Jacobi, A. M., Huang, W., Wang, T., Freimuth, W., Sanz, I., Furie,    R., Mackay, M., Aranow, C., Diamond, B., and Davidson, A. (2010).    Effect of long-term belimumab treatment on B cells in systemic lupus    erythematosus: extension of a phase II, double-blind,    placebo-controlled, dose-ranging study. Arthritis Rheum 62, 201-210.-   Kapoor, P., Ramakrishnan, V., and Rajkumar, S. V. (2012). Bortezomib    combination therapy in multiple myeloma. Semin Hematol 49, 228-242.-   Keyser, F. D. (2011). Choice of Biologic Therapy for Patients with    Rheumatoid Arthritis: The Infection Perspective. Curr Rheumatol Rev    7, 77-87.-   Laskowski, R. A. (2009). PDBsum new things. Nucleic Acids Res 37,    D355-359.-   Mackay, F., Schneider, P., Rennert, P., and Browning, J. (2003).    BAFF AND APRIL: a tutorial on B cell survival. Annu Rev Immunol 21,    231-264.-   Nimmerjahn, F., and Ravetch, J. V. (2008). Fcgamma receptors as    regulators of immune responses. Nat Rev Immunol 8, 34-47.-   Novak, A. J., Darce, J. R., Arendt, B. K., Harder, B., Henderson,    K., Kindsvogel, W., Gross, J. A., Greipp, P. R., and Jelinek, D. F.    (2004). Expression of BCMA, TACl, and BAFF-R in multiple myeloma: a    mechanism for growth and survival. Blood 103, 689-694.-   Queen et al., 1989; WO 90/07861 Patel, D. R., Wallweber, H. J., Yin,    J., Shriver, S. K., Marsters, S. A., Gordon, N. C., Starovasnik,-   M. A., and Kelley, R. F. (2004). Engineering an APRIL-specific B    cell maturation antigen. J Biol Chem 279, 16727-16735.-   Presta, L. G. (2008). Molecular engineering and design of    therapeutic antibodies. Curr Opin Immunol 20, 460-470.-   Raab, M. S., Podar, K., Breitkreutz, I., Richardson, P. G., and    Anderson, K. C. (2009). Multiple myeloma. Lancet 374, 324-339.-   Richardson et al. (2003) New Engl J Med 348:2609-2617.-   Ryan et al (Molecular Cancer Therapeutics, 2007 6(11), 3009)-   Roopenian, D. C., and Akilesh, S. (2007). FcRn: the neonatal Fc    receptor comes of age. Nat Rev Immunol 7, 715-725.-   Suzuki, K. (2013). Current therapeutic strategy for multiple    myeloma. Jpn J Clin Oncol 43, 116-124.-   Thorpe et al., 1982, Immunol. Rev. 62:119-58-   Wang, S. Y., and Weiner, G. (2008). Complement and cellular    cytotoxicity in antibody therapy of cancer. Expert Opin Biol Ther 8,    759-768.-   Woyke et al. (2001) Antimicrob. Agents and Chemother.    45(12):3580-3584

What is claimed is:
 1. A nucleic acid molecule encoding an antibody orantibody fragment, wherein the antibody or antibody fragment comprises:(a) a VH domain that comprises: CDR1 sequence RYWIS (SEQ ID NO: 18) orRYWFS (SEQ ID NO: 19); CDR2 sequence EINPNSSTINYAPSLKDK (SEQ ID NO: 20)or EINPSSSTINYAPSLKDK (SEQ ID NO: 21); and CDR3 sequence SLYYDYGDAYDYW(SEQ ID NO: 22); and (b) a VL domain that comprises: CDR1 sequenceKASQSVX₁X₂NVA (SEQ ID NO: 23), wherein X₁X₂ is ES; CDR2 sequence SASLRFS(SEQ ID NO: 24); and CDR3 sequence QQYNNYPLTFG (SEQ ID NO: 25), whereinsaid antibody or antibody fragment binds an epitope of the extracellulardomain of CD269 (BCMA).
 2. The nucleic acid molecule of claim 1, whereinthe antibody or antibody fragment comprises a VH domain that comprisesthe sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:9.
 3. The nucleic acid molecule of claim 1, wherein the VL domaincomprises the sequence of SEQ ID NO:
 14. 4. The nucleic acid molecule ofclaim 1, wherein the antibody binds an epitope of the N-terminus ofCD269, wherein the epitope consists of amino acids 13, 15, 16, 17, 18,19, 20, 22, 23, 26, 27 or 32 of SEQ ID NO:
 39. 5. The nucleic acidmolecule of claim 1, wherein the antibody binding to CD269 (BCMA)disrupts BAFF-CD269 and/or APRIL-CD269 interaction.
 6. The nucleic acidmolecule of claim 1, wherein the VH domain has at least 80% sequenceidentity to the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 orSEQ ID NO: 9 and wherein the CDR sequences are those recited in claim 1.7. The nucleic acid molecule of claim 1, wherein the VH domain has atleast 90% sequence identity to the sequence of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8 or SEQ ID NO: 9 and wherein the CDR sequences are thoserecited in claim
 1. 8. The nucleic acid molecule of claim 1, wherein thenucleic acid molecule comprises a sequence selected from SEQ ID NO: 33,34 or
 36. 9. A host cell comprising a nucleic acid molecule encoding anantibody or antibody fragment, wherein the antibody or antibody fragmentcomprises: (a) a VH domain that comprises: CDR1 sequence RYWIS (SEQ IDNO: 18) or RYWFS (SEQ ID NO: 19); CDR2 sequence EINPNSSTINYAPSLKDK (SEQID NO: 20) or EINPSSSTINYAPSLKDK (SEQ ID NO: 21); and CDR3 sequenceSLYYDYGDAYDYW (SEQ ID NO: 22); and (b) a VL domain that comprises: CDR1sequence KASQSVX₁X₂NVA (SEQ ID NO: 23), wherein X₁X₂ is ES; CDR2sequence SASLRFS (SEQ ID NO: 24); and CDR3 sequence QQYNNYPLTFG (SEQ IDNO: 25), wherein said antibody or antibody fragment binds an epitope ofthe extracellular domain of CD269 (BCMA).
 10. The host cell of claim 9,wherein the antibody or antibody fragment comprises a VH domain thatcomprises the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 orSEQ ID NO:
 9. 11. The host cell of claim 9, wherein the VL domaincomprises the sequence of SEQ ID NO:
 14. 12. The host cell of claim 9,wherein the antibody binds an epitope of the N-terminus of CD269,wherein the epitope consists of amino acids 13, 15, 16, 17, 18, 19, 20,22, 23, 26, 27 or 32 of SEQ ID NO:
 39. 13. The host cell of claim 9,wherein the antibody binding to CD269 (BCMA) disrupts BAFF-CD269 and/orAPRIL-CD269 interaction.
 14. The host cell of claim 9, wherein the VHdomain has at least 80% sequence identity to the sequence of SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 and wherein the CDRsequences are those recited in claim
 9. 15. The host cell of claim 9,wherein the VH domain has at least 90% sequence identity to the sequenceof SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 and whereinthe CDR sequences are those recited in claim
 9. 16. The host cell ofclaim 9, wherein the nucleic acid molecule comprises a sequence selectedfrom SEQ ID NO: 33, 34 or
 36. 17. The host cell of claim 9, wherein thecell expresses the antibody and the antibody is glycosylated.
 18. Thehost cell of claim 17, wherein the antibody comprises the sequence ofSEQ ID NO: 29, and wherein the antibody comprises a glycan that is anN-linked oligosaccharide chain at Asn297 of the heavy chain consistingof the sequence of SEQ ID NO:
 29. 19. A composition comprising the hostcell of claim 9 and a pharmaceutically acceptable carrier.